Recombinant Subunit Vaccine

ABSTRACT

A method is provided of producing an immunogenic complex comprising a heat shock protein (hsp) coupled to a heterologous antigenic polypeptide, which method comprises: (a) expressing the antigenic polypeptide in a cell which cell has been subjected to a stimulus which causes the induction of a heat shock response in said cells; and (b) recovering the antigenic polypeptide coupled to one or more hsps from said cell or the culture medium. Also provided are immunogenic compositions comprising a heat shock protein (hsp) derived from a non-mammalian eukaryote coupled to a heterologous antigenic polypeptide which composition is capable of inducing an immune response to said antigenic polypeptide in a human or animal.

This application is a divisional of U.S. application Ser. No.10/049,953, filed Feb. 19, 2002, which is a U.S. National Phase ofPCT/AU00/00988, filed Aug. 18, 2000, which claims priority to AustralianPatent Application No. PQ 2337, filed Aug. 19, 1999.

FIELD OF THE INVENTION

The present invention relates generally to the field of therapeutics andthe development thereof for use in animals including mammals, humans,birds and fish. More particularly, it relates to subunit vaccines thatare effective against pathogens causing infections thereof for use inanimals including mammals, humans, birds and fish.

BACKGROUND ART Scientific Background

The development of therapeutics and in particular vaccines directedagainst pathogens such as viruses, bacteria, protozoans, fungi isongoing. Such research has proved invaluable in preventing the spread ofdisease in animals including humans. In fact, in modern medicine,immunotherapy including vaccination has eradicated smallpox andvirtually eradicated diseases such as polio, tetanus, tuberculosis,chicken pox, and measles.

Generally, ideal vaccines have a long shelf life, are capable ofinducing long lasting immunity against a pre-selected pathogen and allof the phenotypic variants, are incapable of causing the disease towhich the vaccine is directed against, are effective therapeutically andprophylactically, are easily prepared using economical standardmethodologies and can be administered easily in the field.

There are four major classes of commercially available vaccines. Theyinclude non-living whole organism vaccines, live attenuated vaccines,vector vaccines, and subunit vaccines. Vaccination with non-livematerials such as proteins generally leads to an antibody response orCD4+ helper T cell response while, vaccination with live materials (e.g.infectious viruses) generally leads to a CD8+ cytotoxic T-lymphocyte(CTL) response. A CTL response is crucial for protection againstpathogens like infectious viruses and bacteria. This poses a practicalproblem, for the only certain way to achieve a CTL response is to uselive agents that are themselves pathogenic. The problem is generallycircumvented by using attenuated viral and bacterial strains or bykilling whole cells that can be used for vaccination. These strategieshave worked well but the use of attenuated strains always carries therisk that the attenuated agent may recombine genetically in the host andturn into a virulent strain. Thus, there is need for therapeutics andmethods that can lead to CD8+ CTL response by vaccination with non-livematerials such as proteins in a specific manner.

Subunit vaccines have provided one means for dealing with some of theseproblems. Such vaccines generally comprise a sub-cellular componentderived from a pathogen of interest. A subunit component can be eitherproduced from a defined sub-cellular fraction of the pathogen, be apurified protein, nucleic acid or a polysaccharide. All of theseelements have an antigenic determinant capable of stimulating an immuneresponse against the pathogen of interest. Generally, the sub-cellularcomponent of the subunit vaccine is obtained either by purifying apreparation of disrupted pathogen or synthesised using well-knownprocedures.

There are, however, several limitations associated with subunitvaccines. First, a requirement for the production of such a vaccine isthat the antigenic determinant(s) must be characterised and identified.This imposes limitations on their use, particularly against highlyvariable antigenic determinants. Second, subunit vaccines are generallyineffective in stimulating cytotoxic T cell responses. Third, theimmunity conferred by subunit vaccines is often short lived andtherefore requires continual booster injections. Very few recombinantexpressed subunit vaccines have been shown to induce strong and longlasting immunity in vaccinated animals (including man). One notableexception is the recombinant surface antigen Hepatitis B vaccine used inman. One of the problems associated with the use of such vaccinesappears to be in correctly presenting the antigens to the immune systemsuch that strong humoral immunity and strong cell-mediated immunity areinduced. In particular, existing recombinant (subunit) vaccines do notappear to result in strong ‘memory’ responses such that vaccinatedanimals react very quickly when they are exposed to natural infectionscaused by a pathogen.

By way of example only, deficiencies in current subunit vaccinesprepared from pestiviruses like bovine viral diarrhoea virus (BVDV) havebeen extensively reported. These studies have shown that even thoughlarge amounts of recombinant protein were used in the vaccines, therewere poor protection rates seen showing that the vaccines failed toprotect from challenge with live BVDV isolates (either homologousprotection or heterologous protection).

The present invention seeks to provide an improved therapeutic vaccinewhich ameliorates at least some of the disadvantages over existing priorart.

General Background

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variation and modifications. The invention alsoincludes all of the steps, features, compositions and compounds referredto or indicated in the specification, individually or collectively, andany and all combinations or any two or more of the steps or features.

Although in general the techniques mentioned herein are well known inthe art, reference may be made in particular to Sambrook et al.,Molecular Cloning, A Laboratory Manual (1989) and Ausubel et al., ShortProtocols in Molecular Biology (1999) 4^(th) Ed, John Wiley & Sons, Inc.

The present invention is not to be limited in scope by the specificembodiments described herein, which are intended for the purpose ofexemplification only. Functionally equivalent products, compositions andmethods are clearly within the scope of the invention as describedherein.

Bibliographic details of the publications referred to in thisspecification are collected at the end of the description. Allreferences cited are hereby incorporated by reference. No admission ismade that any of the references constitute prior art.

Throughout this specification unless the context requires otherwise, theword “comprise”, or variations such as “comprises” or “comprising”, willbe understood to imply the inclusion of a stated integer or group ofintegers but not the exclusion of any other integer or group ofintegers.

SUMMARY OF THE INVENTION

The present invention generally relates to a method of producing animmunogenic composition comprising a heat shock protein (hsp) coupled toa heterologous antigenic polypeptide, which method comprises:

-   -   (a) expressing the antigenic polypeptide in a cell which cell        has been subjected to a stimulus which causes the induction of a        heat shock response in said cells; and    -   (b) recovering the antigenic polypeptide coupled to one or more        hsps from said cell or the culture medium.

Preferably the cell is a non-mammalian eukaryotic cell, more preferablyan insect cell. Typically, the antigenic polypeptide is expressed in thecell by the introduction into the cell of a polynucleotide encoding theantigenic polypeptide operably linked to a regulatory control sequencecapable of directing expression of the polypeptide in the cell.Preferably, the polynucleotide is part of a virus or viral vector, suchas a baculovirus.

The present invention also provides a composition comprising an hspcoupled to a heterologous antigenic polypeptide, the composition beingproduced by the method of the invention. Desirably, the composition iscapable of enhancing the animal's immunocompetence against a pathogen.Preferably the hsp is derived from a non-mammalian eukaryotic cell, morepreferably an insect cell.

Hsps, such as insect hsps, coupled to at least an antigenicpeptide/polypeptide provide an alternative therapeutic vaccine to thosediscussed in the background art, for stimulating an animal's immunesystem to elicit an immune response against foreign pathogens. Whilehsps have been included in therapeutic formulations, no one has, to thebest of the applicant's knowledge, employed hsps from a non-mammalianeukaryote, and more particularly insect cell hsps, coupled to at leastan antigenic peptide/polypeptide in the therapeutic treatment of mammalssuch as domestic animals and humans.

Collective features which different subunit vaccines produced using theinsect cell/baculovirus embodiment of the present invention displayinclude:

-   -   (1) The complex is completely non-infectious.    -   (2) The complex is safe for use in animals since baculoviruses        do not infect animal cells.    -   (3) Therapeutics produced according to the invention will be        cheaper to manufacture in that much higher yields of antigenic        proteins can be produced from baculovirus-infected insect-cell        cultures than from comparable systems.    -   (4) Therapeutics developed according to the invention have been        found to generate very strong memory responses in animals. Thus        when an animal is subsequently challenged with a pathogen they        mount a very rapid and strong response to that pathogen.

Accordingly, the present invention also provides a compositioncomprising a heat shock protein (hsp) derived from a non-mammalianeukaryote coupled to a heterologous antigenic polypeptide whichcomposition is capable of inducing an immune response to said antigenicpolypeptide in a mammal.

Preferably the hsp is an insect heat shock protein. Thus in a preferredembodiment, the present invention provides an improved subunit vaccinecapable of inducing an immune response in an animal comprising: aninsect cell hsps coupled to an antigenic heterologous peptide orpolypeptide.

Preferably, the hsp and the antigenic polypeptide are coupled bynon-covalent means.

Preferably, the antigenic polypeptide is an antigen of a pathogenicorganism, or an antigenic fragment or derivative thereof. Morepreferably the antigenic polypeptide is an antigen of a virus orbacterium, or an antigenic fragment or derivative thereof. In apreferred embodiment, the antigenic polypeptide is derived from apestivirus such as BDVD, more particularly an E1/E2 or NS3/NS4apolypeptide or a fragment or derivative thereof.

In a highly preferred embodiment, the composition is obtainable by amethod comprising:

-   -   (a) expressing the antigenic polypeptide in a non-mammalian        eukaryotic cell which cell has been subjected to a stimulus        which causes the induction of a heat shock response in said        cells; and    -   (b) recovering the antigenic polypeptide coupled to one or more        hsps from said cell or the culture medium.

Thus, the present invention also provides methods for preparing anhsp-antigenic heterologous peptide or polypeptide complex comprising:(a) introducing into a cell a nucleotide sequence encoding at least aantigenic peptide or polypeptide(s), said nucleotide sequence beingintroduced into the cell in such a manner that translation of thenucleotide sequence is possible when the sequence is within the cell;(b) culturing the cell under conditions that provide for expression ofthe peptide or polypeptide; (c) exposing the cell to a stress that iscapable of initiating the production of heat shock proteins in thatcell; and (d) recovering the expressed complex. This procedure can alsobe accompanied by the step of: purifying the complex by any means knownin the art. In a preferred embodiment, the complex produced by themethod is isolated from insect cell polypeptides.

Compositions of the invention are useful in therapeutic methods forinducing an immune response against the antigenic heterologous peptideor polypeptide.

The present invention also provides a pharmaceutical compositioncomprising an immunogenic amount of a composition of the inventiontogether with a pharmaceutically acceptable carrier or diluent.

In a further embodiment the invention provides a method for inducingimmunocompetence in a animal against a pathogen, said method comprisingthe steps of: administering to an animal a therapeutically effectiveamount of a non-mammalian eukaryotic hsp coupled to an antigenic peptideor polypeptide and a pharmaceutically acceptable carrier.

Preferably the methods of the invention comprise methods of eliciting animmune response in an individual in whom the treatment or prevention ofinfectious diseases is desired by administering a composition comprisinga therapeutically effective amount of a complex, in which the complexconsists essentially of hsps non-covalently bound to an antigenicmolecule using any convenient mode of administration. A variety ofadministrative techniques may be utilized, among them oraladministration, nasal and other forms of transmucosal administration,parenteral techniques such as subcutaneous, intravenous andintraperitoneal injections, catheterizations and the like.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the finding that a recombinant subunitvaccine based on BVDV antigens expressed using a baculovirus system ininsect cells that have been heat-shocked is highly effective atpreventing infection of both cows and sheep by BVDV, including achallenge strain which is only distantly related to the BVDV strainwhose polypeptide subunits were used as the basis of the vaccine. Thisis in complete contrast to previously described subunit vaccines againstBVDV which do not afford broad protection against BVDV infection from anumber of different strains.

This system not only results in highly efficacious vaccine compositionbut is also cheaper and safer than the existing alternatives.

Heat Shock Proteins and Antigenic Polypeptides

Heat shock proteins (hsps) are synthesized by a cell in response to heatshock and other forms of cellular stress. The major hsps can accumulateto very high levels in stressed cells, but they occur at low to moderatelevels in cells that are not stressed. Heat shock proteins, useful inthe present invention are proteins (i) whose intracellular concentrationincreases when a cell is exposed to a stressful stimuli, (ii) that arecapable of binding other proteins or peptides, and (iii) are capable ofreleasing the bound proteins or peptides in the presence of adenosinetriphosphate (ATP) or low pH. Particular examples of heat shock proteinssuitable in the context of the present invention include some of theclass of proteins termed molecular chaperones.

Chaperones, including chaperonins, are polypeptides which promoteprotein folding by non-enzymatic means, in that they do not catalyse thechemical modification of any structures in folding polypeptides, butpromote the correct folding of polypeptides by facilitating correctstructural alignment thereof. Molecular chaperones are well known in theart, several families thereof being characterised. Molecular chaperonesare highly conserved between different organisms. Examples of chaperonesinclude the hsp70 family (DnaK type), the hsp60 family (GroEL type),ER-associated chaperones, the hsp90 family, Hsc70, the HSP40 family(DnaJ), mitochondrial hsp70, mitochondrial m-AAA and yeast Ydj1. It isparticularly preferred to use members of the hsp60, hsp70 and hsp90families, whose intracellular concentration rises in response to astress stimulus.

Heat shock proteins are found in prokaryotic cells and eukaryotic cellsand when the immunogenic compositions are produced by the heat shockmethod of the invention, the hsps present in the hsp/antigen complex maybe from any source, prokaryotic or eukaryotic. However, it is preferredthat the hsps are derived from non-mammalian cells, more preferablynon-mammalian eukaryotic cells such as insect cells.

Thus, typically, the hsps present in the compositions of the presentinvention will be derived from non-mammalian eukaryotic cells.

“Non-mammalian eukaryotic cells” are all eukaryotic cells excludingmammalian cells. For example, non-mammalian eukaryotic cells includeyeast cells, fungal cells, invertebrate cells such as insect cells andnon-mammalian vertebrate cells such as amphibian cells. It is preferredto use cells that allow for glycosylation of heterologous polypeptidesexpressed in said cells, and other post-translation modificationstypically performed in the endoplasmic reticulum/golgi body of mammaliancells. However it is not necessary for the cells to carry out preciselythe same post-translational modifications as would be performed in amammalian cell. For example insect cells tend to incorporate lesscomplex sugars into newly synthesized amino acid chains than is the casewith mammalian cells.

“Non-mammalian cells” include the non-mammalian eukaryotic cellsdescribed above as well as prokaryotic cells. Prokaryotic cells includeeubacteria such as E. coli, B. subtilis and any other bacteria suitablefor the expression of heterologous polypeptides. Prokaryotic cells maybe more suited for the expression of bacterial antigens rather thanviral antigens. “Mammalian cells”, which may be used in the method ofthe invention include cell lines such as CHO cells, HeLa cells and anyother mammalian cell type suitable for the expression of heterologouspolypeptides.

The phrase “hsp-antigenic peptide/polypeptide complex”, as used herein,refers to any complex that can be isolated from a culture of cells thatcomprises hsps coupled to at least a heterologous peptide or polypeptidehaving at least one antigenic determinant. Preferably, the coupling isachieved using non-covalent bonding.

The phrase “non-mammalian hsp-antigenic peptide/polypeptide complex”, asused herein, refers to any complex that can be isolated from a cultureof non-mammalian cells that comprises non-mammalian hsps coupled to atleast a heterologous peptide or polypeptide having at least oneantigenic determinant. Preferably, the coupling is achieved usingnon-covalent bonding.

The term “heterologous polypeptide”, as used herein, refers to a peptideor polypeptide not endogenous to the cell, such as the non-mammaliancell, i.e. not encoded by the genome of that cell. Preferably it issomething not normally endogenously complexed with hsps in vivo and doesnot normally co-purify with hsps such as non-mammalian eukaryotic hsps.

The term “polypeptide” as used herein, refers to any amino acid sequencelonger than one amino acid and thus includes peptides of two or moreamino acids in length, typically having more than 5, 10 or 20 aminoacids, which may or may not be modified by chemical means. The term“polypeptide” as used herein also includes proteins, a term whichincludes single-chain polypeptide molecules as well asmultiple-polypeptide complexes where individual constituent polypeptidesare linked by covalent or non-covalent means.

A molecule is “antigenic” when it is capable of specifically interactingwith an antigen recognition molecule of the immune system, such as animmunoglobulin (antibody) or T cell antigen receptor. An antigenicpolypeptide contains at least about 5, and preferably at least about 10,amino acids. An antigenic portion of a molecule can be that portion thatis immunodominant for antibody or T cell receptor recognition, or it canbe a portion used to generate an antibody to the molecule.

Antigenic molecules can be selected from among those known in the art orselected by their ability to bind to antibody or MHC molecules orgenerate immune responses. They include any molecule that will induce animmune response against the infectious agent, e.g., antigens of viruses,bacteria, fungi, parasites etc. In a preferred embodiment of theinvention the antigenic molecules may be derived from, but are notlimited to: (1) viral proteins such as, proteins of any of theimmunodeficiency viruses including human immunodeficiency virus type I(HIV-I) and human immunodeficiency virus type II (HIV-II), flaviviruses,pestiviruses like bovine viral diarrhoea virus (BVDV), border diseasevirus (BDV) and classical swine fever virus (CSFV), hepatitis type A,hepatitis type B, hepatitis type C, hepatitis type E, hepatitis type G(GB), influenza, Varicella, adenovirus, herpes simplex type I (HSV-I),herpes simplex type II (HSV-II), rinderpest, rhinovirus, echovirus,rotavirus, respiratory syncytial virus, papilloma virus, papova virus,cytomegalovirus, echinovirus, arbovirus, hantavirus, coxsackie virus,mumps virus, measles virus, rubella virus and polio virus; (2) antigenicbacterial proteins selected from, but not limited to, mycobacteria,rickettsia, mycoplasma, neisseria and legionella; (3) antigenic protozoaproteins selected from, but not limited to, leishmania, coccidia, andtrypanosoma; and (4) antigenic parasite proteins selected from, but notlimited to, chlamydia and rickettsia.

It is particularly preferred to use antigenic polypeptides derived frompestivirus proteins, such as C/E0, E1/E2, NS3/NS4A and/or NS5A/B.Preferably the pestivirus is selected from BVDV (type 1 and/or type 2)and BDV.

Compositions of the invention may comprise more than one differenthsp/antigenic polypeptide complex. For example, two or more differentantigenic polypeptides may be used to enhance the immunogenicity of theinvention. The two or more antigenic polypeptides may be derived fromthe same protein or from different proteins. It is particularlypreferred in the case of pestiviruses to use at least one antigenicpolypeptide derived from a structural protein and at least one antigenicpolypeptide derived from a non-structural polypeptide. For example, in ahighly preferred embodiment, a composition of the invention comprisesboth an hsp-pestivirus E1/E2 complex and an hsp-pestivirus NS3/NS4Acomplex.

Where more than one antigen is present in a composition of theinvention, at least one antigenic polypeptide should be capable ofproviding a protective immune response when administered to a human oranimal as part of an hsp complex. However, it may be desirable toinclude an hsp/antigenic polypeptide which does not provoke an antibodyresponse and prevents or reduces the generation of an immune response tothat particular antigen when the vaccinated host is subsequentlyinfected by the corresponding natural pathogen. This provides a usefulmarker for vaccinated subjects. By way of example, a truncated BVDVNS3/NS4A antigen has been demonstrated in the Examples section toprovide such a utility.

Preparation of Compositions Comprising hsps Coupled to AntigenicPolypeptides

Compositions of the invention comprising hsps coupled to antigenicpolypeptides may be made by a variety of methods.

For example, purified or partially purified non-mammalian eukaryotichsps obtained by recombinant means, chemical synthesis and/or fromnatural sources such as cell lysates of non-mammalian eukaryotic cells,may be combined in vitro in a suitable vessel with one or more antigenicpolypeptides, which may be obtained by recombinant means, chemicalsynthesis and/or from cell lysates from a suitable natural source, suchas a virally-infected mammalian cell or culture of pathogenic bacteria.The hsps may be pretreated, prior to complexing with an antigenicpolypeptide, with ATP or low pH to remove any peptides that may beassociated with the hsps of interest. Excess ATP may be removed from thepreparation by the addition of apyranase. Where low pH is used, the pHshould be readjusted to neutral pH. Hsps may then be coupled to theantigenic peptide by mixing the pretreated hsp with the antigenicpeptide in a suitable vessel and incubating for from 10 minutes toseveral hours. Typically a ratio of greater than one part antigenicpeptide to one part hsp is used. Optionally, the mixture may then bepurified to remove uncomplexed antigenic peptides.

In a highly preferred embodiment of the present invention, thehsp/antigenic polypeptide complexes are prepared by expressing theantigenic polypeptide in a cell under conditions whereby the stressresponse of the cell is induced and the intracellular levels ofendogenous heat shock proteins is increased. This method is particularlyconvenient since it is not necessary to purify or synthesise hsps, whichis advantageous when cells are used whose hsps are not wellcharacterized. In addition, it likely that the expression of antigenicpolypeptides in the intracellular environment in the presence ofendogenous hsps will lead to more efficient coupling than is possible ina cell-free system.

It is preferred to use non-mammalian cells, such as non-mammalianeukaryotic cells or prokaryotic cells, more preferably non-mammalianeukaryotic cells.

Thus in a preferred method of the invention, a composition of theinvention is prepared by firstly introducing a polynucleotide encodingan antigenic polypeptide of interest into a cell. The polynucleotidewill comprises regulatory control sequences such as a promoter, one ormore enhancers and other transcriptional/translational control sequencesso as to allow for the expression of the antigenic polypeptide in thecell. It may be desirable to use regulatory control sequences that allowfor inducible expression of the antigenic polypeptide, for example inresponse to the administration of an exogenous molecule, or indeed astimulus such as heat shock. This will ensure that synthesis of theantigenic polypeptide does not occur until the levels of heat shockproteins have been upregulated by a heat shock stimulus. Alternatively,temporal control of expression of the antigenic polypeptide may occur byonly introducing the polynucleotide into the cell when it is desired toexpress the polypeptide.

It may also be convenient to include an N-terminal secretion signal sothat the antigenic polypeptide is secreted into the cell medium, as isthe case with the E1/E2 BVDV polypeptide described in the examples.

In a preferred embodiment, the polynucleotide is part of a viral vector,such as a baculovirus vector, or infectious virus, such as abaculovirus. This provides a convenient system since not only canrecombinant viral stocks can be maintained and stored until ready foruse but also the delay in protein expression post-infection is known forviruses such as baculoviruses so the optimum time to shock the hostcells can easily be determined and reproduced. Desirably the nucleotidesequence encoding the antigenic peptide or polypeptides is inserted intoa recombinant baculovirus that has been genetically engineered toproduce antigenic peptide or polypeptides, for instance, by followingthe methods of Smith et al (1983) Mol Cell Biol 12: 2156-2165. A numberof viral transfer vectors allow more than one polynucleotide sequenceencoding a polypeptide to be inserted into the same vector so that theycan be co-expressed by the same recombinant virus.

The method of the invention is not limited to the production of oneantigenic polypeptide at a time in the host cell. Multiplepolynucleotides encoding different antigenic polypeptides of interestmay be introduced into the same host cell. The polynucleotides may bepart of the same nucleic acid molecule or separate nucleic acidmolecules.

Once the polynucleotide encoding the antigenic polypeptide of interesthas been introduced into the host cell, the cell is cultured undersuitable conditions to provide for expression of the protein. The cellsare then heat shocked to induce expression (or enhance expression of)endogenous hsps. Conditions for heat shocking cells are known in the artfor a range of host cells. By way of example, specific conditions forinsect cells are provided below.

Once the cells have been cultured for a suitable period to allow forprotein expression, the antigenic polypeptide/hsp complexes arerecovered from the cell. In this respect, the complexes may be foundwithin the cell and/or in the cell medium. Intracellular complexes maybe recovered using standard lysis and purification procedures. Secretedcomplexes may be recovered from the external medium and purified byprocedures such as concentration using a Centricon tube. When complexesof the present invention are secreted into the medium, it is preferredto adapt the host cells to serum-free medium.

Recovered hsp-/antigenic polypeptide complexes are then typicallycombined with a pharmaceutically acceptable carrier or diluent and/orother components to produce pharmaceutical compositions/vaccinecompositions.

Although a variety of non-mammalian eukaryotic cells, such as yeastcells, fungal cells, invertebrate cells and non-mammalian vertebratecells may be used as host cells according to the methods of the presentinvention, it is preferred to use insect cells. Preferably the insectcells are derived from a Lepidopteran species, e.g. Spodopterafrugiperda such as the Sf9 and Sf21 cell lines.

By way of example only coupling of an antigenic proteins to insect cellhsps in vitro may be accomplished quite simply by placing infected cells(in a container), at between 24 to 48 hrs pest-infection, in a waterbath at, for example, 43° C. for approximately 10 mins to heat shock thecells. The cells are then incubated at about 27.5° C. for a further 2 to24 hrs to allow expression of coupled recombinant protein and hsps. Atthe end of 72 to 96 hrs in total post-infection, harvesting of therecombinant protein cultures is carried out.

The optimum periods and temperatures for inducing a heat shock responsein various suitable host cells and allowing for optimum expression ofthe antigenic polypeptides can easily be determined by the skilledperson, for example by conducting a time course, or have already beendetermined for many cell lines.

Typically, the heat shock response is induced after induction of theexpression of the antigenic polypeptide or at about the same time, morepreferably after.

Hsp/antigenic polypeptide compositions may optionally be tested forimmunogenicity prior to administration to human or animal subjects usingin vitro assays known in the art such as the mixed lymphocyte targetculture assay (MLTC).

Compositions

Compositions of the invention comprising hsps coupled to antigenicpolypeptides may preferably be combined with various components toproduce compositions of the invention. Preferably the compositions arecombined with a pharmaceutically acceptable carrier or diluent toproduce a pharmaceutical composition (which may be for human or animaluse). Compositions of the invention comprising hsps coupled to antigenicpolypeptides may also be combined with suitable components to obtainvaccine compositions.

Thus, the invention provides pharmaceutical/vaccine compositionscomprising a non-mammalian eukaryotic hsp-antigenic peptide orpolypeptide complex that enhances the immunocompetence of the hostindividual and elicits specific immunity against pathogens. Thetherapeutic regimens and pharmaceutical compositions of the inventionare described below. These compositions are believed to have thecapacity to prevent the onset and progression of infectious diseases.

Generally pharmaceutical compositions and/or vaccine compositions of theinvention will comprise a therapeutically effective amount of an hspcoupled to an antigenic polypeptide.

The phrase “pharmaceutically acceptable carrier or diluent” refers tomolecular entities and compositions that are physiologically tolerableand do not typically produce an allergic or similarly untoward reaction,such as gastric upset, dizziness and the like, when administered to ahuman. Preferably, as used herein, the term “pharmaceuticallyacceptable” means approved by a regulatory agency of the federal or astate government or listed in the U.S. Pharmacopeia or other generallyrecognized pharmacopeia for use in animals, and more particularly inhumans. The term “carrier” refers to a diluent, adjuvant, excipient, orvehicle with which the compound is administered. Such pharmaceuticalcarriers can be sterile liquids, such as water and oils, including thoseof petroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil and the like. Water or solublesaline solutions and aqueous dextrose and glycerol solutions arepreferably employed as carriers, particularly for injectable solutions.Suitable pharmaceutical carriers are described in Martin, Remington'sPharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, Pa.,(1990).

The phrase “therapeutically effective amount” as used herein refers toan amount sufficient to stimulate by at least about 15%, preferably byat least 50%, more preferably by at least 90%, and most preferablycompletely, a animals immune system causing it to generate animmunological memory against the antigenic determinant.

In general, comprehended by the invention are pharmaceuticalcompositions comprising therapeutically effective amounts of theinvention together with pharmaceutically acceptable diluents,preservatives, solubilizers, emulsifiers, adjuvants and/or carriers.Such compositions may include diluents of various buffer content (e.g.,Tris-HCl, acetate, phosphate), pH and ionic strength; additives such asdetergents and solubilizing agents (e.g., Tween 80, Polysorbate 80),anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives(e.g., Thimersol, benzyl alcohol) and bulking substances (e.g., lactose,mannitol); incorporation of the material into particulate preparationsof polymeric compounds such as polylactic acid, polyglycolic acid, etc.or into liposomes. Hylauronic acid may also be used. Such compositionsmay influence the physical state, stability, rate of in vivo release,and rate of in vivo clearance of the present complexes. See, e.g.,Martin, Remington's Pharmaceutical Sciences, 18th Ed. (1990, MackPublishing Co., Easton, Pa. 18042) pages 1435-1712 which are hereinincorporated by reference. The compositions may be prepared in liquidform, or may be in dried powder, such as lyophilized form.

Pharmaceutical compositions may be for administration by injection, orprepared for oral, pulmonary, nasal or other forms of administration.The mode of administration of the complexes prepared in accordance withthe invention will necessarily depend upon such factors as the stabilityof the complex under physiological conditions, the intensity of theimmune response required, the type of pathogen etc.

Preferably, the complex is administered using standard procedures, forexample, intravenously, subcutaneously, intramuscularly, intraorbitally,ophthalmically, intraventricularly, intracranially, intracapsularly,intraspinally, intracisternally, intraperitoneally, buccal, rectally,vaginally, intranasally, orally or by aerosol administration.

Vaccines may also be prepared from one or more hsp/antigenic polypeptidecomplexes of the invention. The preparation of vaccines which containimmunogenic complexes as active ingredients, is known to one skilled inthe art. Typically, such vaccines are prepared as injectables, either asliquid solutions or suspensions; solid forms suitable for solution in,or suspension in, liquid prior to injection may also be prepared. Thepreparation may also be emulsified, or the protein encapsulated inliposomes. The active immunogenic ingredients are often mixed withexcipients which are pharmaceutically acceptable and compatible with theactive ingredient. Suitable excipients are, for example, water, saline,dextrose, glycerol, ethanol, or the like and combinations thereof.

In addition, if desired, the vaccine may contain minor amounts ofauxiliary substances such as wetting or emulsifying agents, pH bufferingagents, and/or adjuvants which enhance the effectiveness of the vaccine.

The term “adjuvant” as used herein, refers to a compound or mixture thatenhances the immune response to a composition-containing an hsp coupledto a peptide or polypeptide having at least one antigenic determinant.An adjuvant can serve as a tissue depot that slowly releases the antigenand also as a lymphoid system activator that non-specifically enhancesthe immune response.

Examples of adjuvants which may be effective include but are not limitedto: aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine(thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637,referred to as nor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(CGP 19835A, referred to as MTP-PE), and RIBI, which contains threecomponents extracted from bacteria, monophosphoryl lipid A, trehalosedimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween80 emulsion.

Further examples of adjuvants and other agents include aluminumhydroxide, aluminum phosphate, aluminum potassium sulfate (alum),beryllium sulfate, silica, kaolin, carbon, water-in-oil emulsions,oil-in-water emulsions, muramyl dipeptide, bacterial endotoxin, lipid X,Corynebacterium parvum (Propionobacterium acnes), Bordetella pertussis,polyribonucleotides, sodium alginate, lanolin, lysolecithin, vitamin A,saponin, immuno stimulating complexes (ISCOMs), liposomes, levamisole,DEAE-dextran, blocked copolymers or other synthetic adjuvants. Suchadjuvants are available commercially from various sources, for example,Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.) or Freund'sIncomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit,Mich.).

Typically, adjuvants such as Amphigen (oil-in-water), Alhydrogel(aluminum hydroxide), or a mixture of Amphigen and Alhydrogel are used.Only aluminum hydroxide is approved for human use.

The proportion of immunogen and adjuvant can be varied over a broadrange so long as both are present in effective amounts. For example,aluminum hydroxide can be present in an amount of about 0.5% of thevaccine mixture (Al₂O₃ basis). Conveniently, the vaccines are formulatedto contain a final concentration of immunogen in the range of from 0.2to 200 μg/ml, preferably 5 to 50 μg/ml, most preferably 15 μg/ml.

After formulation, the vaccine may be incorporated into a sterilecontainer which is then sealed and stored at a low temperature, forexample 4° C., or it may be freeze-dried. Lyophilisation permitslong-term storage in a stabilised form.

The effectiveness of an adjuvant may be determined by measuring theamount of antibodies directed against an immunogenic complex of theinvention resulting from administration of this complex in vaccineswhich are also comprised of the various adjuvants.

The vaccines are conventionally administered parenterally, by injection,for example, either subcutaneously or intramuscularly. Additionalformulations which are suitable for other modes of administrationinclude suppositories and, in some cases, oral formulations. Forsuppositories, traditional binders and carriers may include, forexample, polyalkylene glycols or triglycerides; such suppositories maybe formed from mixtures containing the active ingredient in the range of0.5% to 10%, preferably 1% to 2%. Oral formulations include suchnormally employed excipients as, for example, pharmaceutical grades ofmannitol, lactose, starch, magnesium stearate, sodium saccharine,cellulose, magnesium carbonate, and the like. These compositions takethe form of solutions, suspensions, tablets, pills, capsules, sustainedrelease formulations or powders and contain 10% to 95% of activeingredient, preferably 25% to 70%. Where the vaccine composition islyophilised, the lyophilised material may be reconstituted prior toadministration, e.g. as a suspension. Reconstitution is preferablyeffected in buffer

Capsules, tablets and pills for oral administration to a patient may beprovided with an enteric coating comprising, for example, Eudragit “S”,Eudragit “L”, cellulose acetate, cellulose acetate phthalate orhydroxypropylmethyl cellulose.

The complexes of the invention may be formulated into the vaccine asneutral or salt forms. Pharmaceutically acceptable salts include theacid addition salts (formed with free amino groups of the peptide) andwhich are formed with inorganic acids such as, for example, hydrochloricor phosphoric acids, or such organic acids such as acetic, oxalic,tartaric and maleic. Salts formed with the free carboxyl groups may alsobe derived from inorganic bases such as, for example, sodium, potassium,ammonium, calcium, or ferric hydroxides, and such organic bases asisopropylamine, trimethylamine, 2-ethylamino ethanol, histidine andprocaine.

Other Active Ingredients

Compositions of the present invention may further comprise antigenicpolypeptides that are not coupled to hsps and/or biologically activemolecules whose primary purpose is not to serve as an antigen but tomodulate the immune response in some other aspect. Examples ofbiologically molecules that modulate the immune system of an animal orhuman subject include cytokines.

The term “cytokine” refers to any secreted polypeptide that influencesthe function of other cells mediating an immune response. Some examplesof cytokines include, but are not limited to, interleukin-1.alpha.(IL-1.alpha.), interleukin-1.beta. (IL-1.beta.), interleukin-2 (IL-2),interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5),interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8),interleukin-9 (IL-9), interleukin-10 (IL-10), interleukin-11 (IL-11),interleukin-12 (IL-12), interferon .alpha. (IFN.alpha.), interferon.beta. (IFN.beta.), interferon .gamma. (IFN.gamma.), tumor necrosisfactor alpha. (TNF.varies.), tumor necrosis factor beta. (TNF.beta.),granulocyte colony stimulating factor (G-CSF), granulocyte/macrophagecolony stimulating factor (GM-CSF), and transforming growth factor.beta. (TGF-.beta.).

Therapeutic Uses

The compositions of the present invention may be used to vaccinateanimals and humans against infectious diseases. The term “animal”includes: mammals such as farm animals including sheep, goats, pigs,cows, horses, llamas, household pets such as dogs and cats, andprimates; birds, such as chickens, geese and ducks; fish; and reptilessuch as crocodiles and alligators.

Thus, the present invention a method of inducing a protective immuneresponse in an animal or human against a pathogen, which methodcomprises administering to said animal or human an effective amount of acomposition of the invention.

Thus, the present invention also provides methods for enhancing ananimal's immunocompetence and the activity of its immune effector cellsagainst a pathogen. Such methods will typically include the step of:administering a composition comprising a therapeutically effectiveamount of an insect cell hsp-antigenic peptide/polypeptide complex, inwhich the complex consists essentially of an hsp coupled to anheterologous peptide or polypeptide antigenic molecule.

In a highly preferred embodiment, the present invention provides hspscomplexes prepared from proteins and polypeptides derived from apestivirus, preferably bovine viral diarrhoea virus (BVDV), morepreferably BVDV E1/E2 and/or NS3/NS4A and/or antigenic fragmentsthereof. The hsp may be derived from any source but is preferablyderived from a non-mammalian eukaryotic cell.

The term “vaccine” as used herein, refers to mean any composition of theinvention containing an hsp coupled to a peptide or polypeptide havingat least one antigenic determinant which when administered to a animalis capable of stimulating an immune response against the antigenicdeterminant. It will be understood that the term vaccine does notnecessarily imply that the composition will provide a completeprotective response. Rather a therapeutic effect will be sufficient.

The phrase “immune response” refers to any cellular process that isproduced in the animal following stimulation with an antigen and isdirected toward the elimination of the antigen from the animal. Theimmune response typically is mediated by one or more populations ofcells characterized as being lymphocytic and/or phagocytic in nature.

The immune response generated against an introduced hsps-antigenicpeptide or polypeptide complex will be dictated by the amino acidconstitution of the antigenic determinants located on the peptide orpolypeptide in the complex. Such determinants may define either humoralor cell mediated antigenic regions. Without being limited to anyparticular mode of action, it is contemplated that the immune responsegenerated by the insect cell hsps-antigenic peptide or protein complexwill preferably include both humoral and cell mediated immune responses.Where a cell mediated immune response is effected it preferably leads toa T cell cascade, and more specifically by means of a cytotoxic T cellcascade.

The term “cytotoxic T cell”, as used herein, refers to any T lymphocyteexpressing the cell surface glycoprotein marker CD8+ that is capable oftargeting and lysing a target cell which bears a majorhistocompatibility class I (MHC Class I) complex on its cell surface andis infected with an intracellular pathogen.

Diseases that might be treated or prevented by the methods of thepresent invention are caused by pathogens including, but not limited toviruses, bacteria, fungi, protozoa and parasites.

Viral diseases that can be treated or prevented by the methods of thepresent invention include, but are not limited to, those caused byimmunodeficiency viruses including human immunodeficiency virus type I(HIV-I) and human immunodeficiency virus type II (HIV-II), flaviviruses,hepatitis type A, hepatitis type B, hepatitis type C, pestiviruses likebovine viral diarrhoea virus (BVDV), Border Disease Virus (BDV) andclassical swine fever virus (CSFV), influenza, Varicella, adenovirus,herpes simplex type I (HSV-I), herpes simplex type II (HSV-II),rinderpest, rhinovirus, echovirus, rotavirus, respiratory syncytialvirus, papilloma virus, papova virus, cytomegalovirus, echinovirus,arbovirus, hantavirus, coxsackie virus, mumps virus, measles virus,rubella virus and polio virus.

Bacterial diseases that can be treated or prevented by the methods ofthe present invention are caused by bacteria including, but not limitedto, mycobacteria, rickettsia, mycoplasma, neisseria and legionella.

Protozoal diseases that can be treated or prevented by the methods ofthe present invention are caused by protozoa including, but not limitedto, leishmania, coccidia, and trypanosoma.

Parasitic diseases that can be treated or prevented by the methods ofthe present invention are caused by parasites including, but not limitedto, chlamydia and rickettsia.

According to a highly preferred embodiment there is provided a subunittherapeutic against BVDV infections in cattle herds that is capable ofinducing an immune response in cattle comprising: an hsp coupled to anantigenic BVDV peptide or polypeptide. Preferably, the hsps arenon-covalently coupled to the antigenic BVDV peptide or polypeptide(s).

The primary aim of all modern pestivirus therapeutics is based on theirability to prevent the transplacental transmission of the virus thusbreaking the cycle of infection in cattle herds. The foetal protectionindex is the only objective measurement of efficacy of BVDV vaccines. Ithas increasingly been adopted overseas as the demonstrated requirementfor future BVDV vaccine registrations.

A surprising feature of this embodiment of the invention is that 100%protection has been observed using the vaccine prior to BVDV infectionsin cattle. Such protection has resulted from the unusually high levelsof neutralising antibodies to BVDV after only a short exposure to thelive virus challenge. The subunit vaccine provides levels ofneutralising antibodies never observed before by the applicants. Inaddition, the subunit vaccine provides a surprisingly effective memorycytotoxic T cell response. Further, the subunit vaccine provides for thefirst time a 100% effective inhibition of transplacental transfer ofvirus from dam to foetus.

In addition to the above, vaccines produced in accordance with thisembodiment of the invention have particular advantages over other BVDVvaccines and in particular BVDV subunit vaccines. One of the drawbacksof using live attenuated vaccines or whole organism vaccines is thelikelihood of infection of cell cultures during manufacture of thevaccine. In contrast, subunit vaccines are non-infectious. Inparticular, the subunit vaccine of the invention does not require serumfor manufacture, thus alleviating the risks involved with handling serumproducts such as foetal calf serum.

Another advantage of these vaccines is their safety when vaccinatingcommercial herds of animals such as cattle against BVDV. The subunitvaccine of the present invention can be used safely in animals withoutthe risk of infection (live attenuated viruses) or infection of theanimal cells with the virus. Thus it is safe to use in all animals,including pregnant animals.

A further advantage of BVDV subunit vaccines produced in accordance withthe invention is the cost-effective nature of producing the subunitvaccine. It is possible to obtain high yields of antigenic proteins. Inparticular, in a preferred embodiment of the invention a subunit vaccineis produced using a vector baculovirus to infect insect cell cultures,producing a subunit vaccine effective against BVDV. The resultingvaccine has efficacy against a much wider range of antigenically diverseBVDV isolates.

Any antigenic region from BVDV may be used in the identified subunitvaccines. Preferably, the antigenic peptide or polypeptides are derivedfrom the major immunogenic regions E0, E1/E2 and NS3/NS4A. In a highlypreferred form of this embodiment of the invention the subunit vaccineis produced using a truncated NS3/NS4A protein from isolates of BVDV.Surprisingly, this NS3/NS4A protein antigen does not cause theproduction of a detectable range of antibodies in the serum of cattlevaccinated with the subunit vaccine. Thus the incorporation of theNS3/NS4A protein into the subunit vaccine provides a useful marker todistinguish infected cattle within a herd from vaccinated cattle. It isthe preferred practice in Europe and the US to include a marker in thevaccine, identifying infected animals from vaccinated animals. Thus thesubunit vaccine of the present invention provides an excellent markerfor distinguishing infected animals within a herd from vaccinatedanimals.

Administration

Parenteral Delivery

The compounds provided herein can be formulated into pharmaceuticalcompositions by admixture with pharmaceutically acceptable nontoxicexcipients and carriers and administered by any parenteral techniquessuch as subcutaneous, intravenous and intraperitoneal injections. Inaddition the formulations may optionally contain one or more adjuvants.

Oral Delivery

Contemplated for use herein are oral solid dosage forms, which aredescribed generally in Martin, Remington's Pharmaceutical Sciences, 18thEd. (1990 Mack Publishing Co. Easton Pa. 18042) at Chapter 89, which isherein incorporated by reference. Solid dosage forms include tablets,capsules, pills, troches or lozenges, cachets or pellets. Also,liposomal or proteinoid encapsulation may be used to formulate thepresent compositions (as, for example, proteinoid microspheres reportedin U.S. Pat. No. 4,925,673). Liposomal encapsulation may be used and theliposomes may be derivatised with various polymers (E.g., U.S. Pat. No.5,013,556). A description of possible solid dosage forms for thetherapeutic is given by Marshall, in Modern Pharmaceutics, Chapter 10,Banker and Rhodes ed., (1979), herein incorporated by reference. Ingeneral, the formulation will include the hsp-antigenicpeptide/polypeptide complex (or a chemically modified form thereof), andinert ingredients which allow for protection against the stomachenvironment, and release of the biologically active material in theintestine.

Also specifically contemplated are oral dosage forms of the abovederivatised hsp-antigenic peptide/polypeptide complexes. In this respectthe complexes may be chemically modified so that oral delivery isefficacious. Generally, the chemical modification contemplated is theattachment of at least one moiety to the protein (or peptide) moleculeitself, where said moiety permits (a) inhibition of proteolysis; and (b)uptake into the blood stream from the stomach or intestine. Also desiredis the increase in overall stability of the protein and increase incirculation time in the body. Examples of such moieties include:polyethylene glycol, copolymers of ethylene glycol and propylene glycol,carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone and polyproline. Abuchowski et al., 1981, supra; Newmark etal., J. Appl. Biochem., 4:185-189 (1982). Other polymers that could beused are poly-1,3-dioxolane and poly-1,3,6-tioxocane. Preferred forpharmaceutical usage, as indicated above, are polyethylene glycolmoieties.

For the hsp-antigenic peptide/polypeptide complex the location ofrelease may be the stomach, the small intestine (the duodenum, thejejunem, or the ileum), or the large intestine. One skilled in the arthas available formulations that will not dissolve in the stomach, yetwill release the material in the duodenum or elsewhere in the intestine.Preferably, the release will avoid the deleterious effects of thestomach environment, either by protection of the complex or by releaseof the biologically active material beyond the stomach environment, suchas in the intestine.

To ensure full gastric resistance, a coating impermeable to at least pH5.0 is essential. Examples of the more common inert ingredients that areused as enteric coatings are cellulose acetate trimellitate (CAT),hydroxypropylmethylcellulose phthalate (HPMCP), HPMCP 50, HPMCP 55,polyvinyl acetate phthalate (PVAP), Eudragit L30D, Aquateric, celluloseacetate phthalate (CAP), Eudragit L, Eudragit S, and Shellac. Thesecoatings may be used as mixed films.

A coating or mixture of coatings can also be used on tablets, which arenot intended for protection against the stomach. This can include sugarcoatings, or coatings which make the tablet easier to swallow. Capsulesmay consist of a hard shell (such as gelatin) for delivery of drytherapeutic i.e. powder; for liquid forms, a soft gelatin shell may beused. The shell material of cachets could be thick starch or otheredible paper. For pills, lozenges, molded tablets or tablet triturates,moist massing techniques can be used.

The therapeutic can be included in the formulation as finemultiparticulates in the form of granules or pellets of particle sizeabout 1 mm. The formulation of the material for capsule administrationcould also be as a powder, lightly compressed plugs or even as tablets.The therapeutic could be prepared by compression.

Colorants and flavoring agents may all be included. For example, thehsp-antigenic peptide/polypeptide complex may be formulated (such as byliposome or microsphere encapsulation) and then further contained withinan edible product, such as a refrigerated beverage containing colorantsand flavoring agents.

One may dilute or increase the volume of the therapeutic with an inertmaterial. These diluents could include carbohydrates, especiallymannitol, alpha-lactose, anhydrous lactose, cellulose, sucrose, modifieddextrans and starch. Certain inorganic salts may be also be used asfillers including calcium triphosphate, magnesium carbonate and sodiumchloride. Some commercially available diluents are Fast-Flo, Emdex,STA-Rx 1500, Emcompress and Avicell.

Disintegrants may be included in the formulation of the therapeutic intoa solid dosage form. Materials used as disintegrants include but are notlimited to starch including the commercial disintegrant based on starch,Explotab. Sodium starch glycolate, Amberlite, sodiumcarboxymethylcellulose, ultramylopectin, sodium alginate, gelatin,orange peel, acid carboxymethyl cellulose, natural sponge and bentonitemay all be used. Another form of the disintegrants are the insolublecationic exchange resins. Powdered gums may be used as disintegrants andas binders and these can include powdered gums such as agar, Karaya ortragacanth. Alginic acid and its sodium salt are also useful asdisintegrants.

Binders may be used to hold the therapeutic agent together to form ahard tablet and include materials from natural products such as acacia,tragacanth, starch and gelatin. Others include methyl cellulose (MC),ethyl cellulose (EC) and carboxymethyl cellulose (CMC). Polyvinylpyrrolidone (PVP) and hydroxypropylmethyl cellulose (HPMC) could both beused in alcoholic solutions to granulate the therapeutic.

An antifrictional agent may be included in the formulation of thetherapeutic to prevent sticking during the formulation process.Lubricants may be used as a layer between the therapeutic and the diewall, and these can include but are not limited to: stearic acidincluding its magnesium and calcium salts, polytetrafluoroethylene(PTFE), liquid paraffin, vegetable oils and waxes. Soluble lubricantsmay also be used such as sodium lauryl sulphate, magnesium laurylsulphate, polyethylene glycol of various molecular weights, and Carbowax4000 and 6000.

Glidants that might improve the flow properties of the complex duringformulation and to aid rearrangement during compression might be added.The glidants may include starch, talc, pyrogenic silica and hydratedsilicoaluminate.

To aid dissolution of the therapeutic into the aqueous environment, asurfactant might be added as a wetting agent. Surfactants may includeanionic detergents such as sodium lauryl sulphate, dioctyl sodiumsulphosuccinate and dioctyl sodium sulfonate. Cationic detergents mightbe used and could include benzalkonium chloride or benzethomiumchloride. The list of potential nonionic detergents that could beincluded in the formulation as surfactants are lauromacrogol 400,polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and60, glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fattyacid ester, methyl cellulose and carboxymethyl cellulose. Thesesurfactants could be present in the formulation of the complex eitheralone or as a mixture in different ratios.

Additives which potentially enhance uptake of the complex are forinstance the fatty acids oleic acid, linoleic acid and linolenic acid.

Controlled release formulation may be desirable. The complex could beincorporated into an inert matrix which permits release by eitherdiffusion or leaching mechanisms i.e., gums. Slowly degeneratingmatrices may also be incorporated into the formulation. Another form ofa controlled release of this therapeutic is by a method based on theOros therapeutic system (Alza Corp.), i.e. the drug is enclosed in asemipermeable membrane which allows water to enter and push drug outthrough a single small opening due to osmotic effects. Some entericcoatings also have a delayed release effect.

Other coatings may be used for the formulation. These include a varietyof sugars which could be applied in a coating pan. The therapeutic agentcould also be given in a film-coated tablet; the materials used in thisinstance are divided into 2 groups. The first are the nonentericmaterials and include methyl cellulose, ethyl cellulose, hydroxyethylcellulose, methylhydroxy-ethyl cellulose, hydroxypropyl cellulose,hydroxypropyl-methyl cellulose, sodium carboxy-methyl cellulose,providone and the polyethylene glycols. The second group consists of theenteric materials that are commonly esters of phthalic acid.

A mix of materials might be used to provide the optimum film coating.Film coating may be carried out in a pan coater or in a fluidized bed orby compression coating.

Pulmonary Delivery

Also contemplated herein is pulmonary delivery of the complex. Thehsp-antigenic peptide/polypeptide complex may be delivered to the lungsof an animal while inhaling and traverses across the lung epitheliallining to the blood-stream.

Contemplated for use in the practice of this invention are a wide rangeof mechanical devices designed for pulmonary delivery of therapeuticproducts, including but not limited to nebulizers, metered-doseinhalers, and powder inhalers, all of which are familiar to thoseskilled in the art.

Some specific examples of commercially available devices suitable forthe practice of this invention are the Ultravent nebulizer, manufacturedby Mallinckrodt, Inc., St. Louis, Mo.; the Acorn II nebulizer,manufactured by Marquest Medical Products, Englewood, Colo.; theVentolin metered dose inhaler, manufactured by Glaxo Inc., ResearchTriangle Park, N.C.; and the Spinhaler powder inhaler, manufactured byFisons Corp., Bedford, Mass.

All such devices require the use of formulations suitable for thedispensing of the complex. Typically, each formulation is specific tothe type of device employed and may involve the use of an appropriatepropellant material, in addition to the usual diluents, adjuvants and/orcarriers useful in therapy. Also, the use of liposomes, microcapsules ormicrospheres, inclusion complexes, or other types of carriers iscontemplated. Chemically modified protein may also be prepared indifferent formulations depending on the type of chemical modification orthe type of device employed.

Formulations suitable for use with a nebulizer, either jet orultrasonic, will typically comprise the complex suspended in water at aconcentration of about 0.1 to 25 mg of biologically active protein perml of solution. The formulation may also include a buffer and a simplesugar (e.g., for protein stabilization and regulation of osmoticpressure). The nebulizer formulation may also contain a surfactant, toreduce or prevent surface induced aggregation of the protein caused byatomization of the solution in forming the aerosol.

Formulations for use with a metered-dose inhaler device will generallycomprise a finely divided powder containing the complex suspended in apropellant with the aid of a surfactant. The propellant may be anyconventional material employed for this purpose, such as achlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or ahydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane,dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, orcombinations thereof. Suitable surfactants include sorbitan trioleateand soya lecithin. Oleic acid may also be useful as a surfactant.

Formulations for dispensing from a powder inhaler device will comprise afinely divided dry powder containing the complex and may also include abulking agent, such as lactose, sorbitol, sucrose, or mannitol inamounts which facilitate dispersal of the powder from the device, e.g.,50 to 90% by weight of the formulation. The protein (or derivative)should most advantageously be prepared in particulate form with anaverage particle size of less than 10 microns, most preferably 0.5 to 5microns, for most effective delivery to the distal lung.

Nasal Delivery

Nasal delivery of the complex is also contemplated. Nasal deliveryallows the passage of the protein to the blood stream directly afteradministering the therapeutic product to the nose, without the necessityfor deposition of the product in the lung. Formulations for nasaldelivery include those with dextran or cyclodextran.

Administration with Other Compounds

The therapeutic regimens and pharmaceutical compositions of theinvention may be coadministered with additional immune responseenhancers or biological response modifiers including, but not limitedto, the cytokines IFN-.alpha., IFN-.gamma., IL-2, IL-4, IL-6, TNF, orother cytokine-affecting immune cells. In accordance with this aspect ofthe invention, the complexes of the hsp and antigenic molecule areadministered in combination therapy with a therapeutically active amountof one or more of these cytokines. As used herein, the term “cytokine”is meant to mean any secreted polypeptide that influences the functionof other cells mediating an immune response. Accordingly, it iscontemplated that the complex can be coadministered with a cytokine toenhance the immune response directed against the tumor. Preferredcytokines include, but are not limited to, interleukin-1.alpha.(IL-1.alpha.), interleukin-1.beta. (IL-1.beta.), interleukin-2 (IL-2),interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5),interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8),interleukin-9 (IL-9), interleukin-10 (IL-10), interleukin-11 (IL-11),interleukin-12 (IL-12), interferon .alpha. (IFN.alpha.), interferonbeta. (IFN.beta.), interferon gamma. (IFN.gamma.), tumor necrosis factoralpha. (TNF.varies.), tumor necrosis factor .beta. (TNF.beta.),granulocyte colony stimulating factor (G-CSF), granulocyte/macrophagecolony stimulating factor (GM-CSF), and transforming growth factor beta.(TGF-.beta.).

In addition, conventional antibiotics may be coadministered with thestress protein-peptide complex. The choice of suitable antibiotics willhowever be dependent upon the disease in question.

Dosages

For all of the above molecules, as further studies are conducted,information will emerge regarding appropriate dosage levels fortreatment of various conditions in various patients, and the ordinaryskilled worker, considering the therapeutic context, age and generalhealth of the recipient, will be able to ascertain the proper dosage.

Typically, the complex should be administered in an amount sufficient toinitiate in the animal an immune response against the pathogen followingsubsequent challenge. The amount of insect cell hsp-antigenicpeptide/polypeptide complex administered preferably is in the range ofabout 0.1-1.0 micrograms of complex/kg body weight of theanimal/administration, and most preferably about 0.2 to 0.5 microgramsof complex/kg body weight of the animal/administration.

It is contemplated that a typical dose will be in the range of about 0.5to about 50 micrograms for a human subject weighing about 75 kg. Inaddition, it is contemplated that the strength of the immune responsemay be enhanced by repeatedly administering the complex to theindividual. Thus in one example the animal may receive at least twodoses of the insect cell hsp-antigenic peptide/polypeptide complex atapproximately monthly intervals. If necessary, the immune response maybe boosted at a later date by subsequent administration of the complex.It is contemplated, however, that the optimal dosage and immunizationregimen may be found by routine experimentation by one skilled in theart.

Kits

The invention also provides kits for carrying out the therapeuticregimens of the invention. Such kits comprise in one or more containerstherapeutically or prophylactically effective amounts of the insect cellhsp-antigenic peptide/polypeptide complex in pharmaceutically acceptableform. The hsp-antigenic molecule complex in a vial of a kit of theinvention may be in the form of a pharmaceutically acceptable solution,e.g., in combination with sterile saline, dextrose solution, or bufferedsolution, or other pharmaceutically acceptable sterile fluid.Alternatively, the complex may be lyophilized or desiccated; in thisinstance, the kit optionally further comprises in a container apharmaceutically acceptable solution (e.g., saline, dextrose solution,etc.), preferably sterile, to reconstitute the complex to form asolution for injection purposes.

In another embodiment, a kit of the invention further comprises a needleor syringe, preferably packaged in sterile form, for injecting thecomplex, and/or a packaged alcohol pad. Instructions are optionallyincluded for administration of hsp-antigenic molecule complexes by aclinician or by the patient.

BEST MODE(S) FOR CARRYING OUT THE INVENTION

Further features of the present invention are more fully described inthe following non-limiting Figures and Examples. It is to be understood,however, that this description is included solely for the purposes ofexemplifying the present invention. It should not be understood in anyway as a restriction on the broad description of the invention as setout above. In the drawings:

FIG. 1 The development of anti-E2 (neutralising) antibody concentrationin both vaccinated (-∘-) and control (-▪-) groups of heifers before andafter live virus challenge (C).

FIG. 2 The development of anti-NS3 antibody concentration in both thevaccinated (-∘-) and control (-▪-) groups of heifers before and aftervirus challenge (C).

FIG. 3 The development of anti-E2 (neutralising) antibody concentrationin both vaccinated (-∘-) and control (-▪-) groups of sheep before andafter live virus challenge (C).

FIG. 4 The development of anti-NS3 antibody concentration in both thevaccinated (-∘-) and control (-▪-) groups of sheep before and aftervirus challenge (C).

Methods of molecular cloning, and protein chemistry methods that are notexplicitly described in the following Examples are reported in theliterature and are known by those skilled in the art. General texts thatdescribed conventional molecular biology, microbiology, and recombinantDNA techniques within the skill of the art, included, for example:Sambrook et al. (1989); Glover (1985); and Ausubel, et al. Currentprotocols in molecular biology.

EXAMPLE 1 Production of Recombinant Baculoviruses and Expression ofRecombinant Pestivirus Proteins Pestiviruses

Pestivirus isolates Trangie-D10, Bega and Clover Lane were isolated andcharacterised at the Virology Department, Elizabeth MacarthurAgricultural Institute (EMAI). The BVDV isolates Trangie D10 and Begarepresent Bovine Viral Diarrhoea Virus (BVDV) Type 1 pestiviruses, whilethe Clover Lane isolate is a Border Disease Virus (BDV) isolate.

The GenBank accession numbers of the Australian virus isolates used inthe subunit vaccine and the relative positions of the genomic fragmentsused relative to the reference strain BVDV NADL (Accession numberM31182) were as follows: Bega (AF049221) E0 partial sequence codesrelative to the whole virus genome from 1171-1897; Trangie (AF049222) E0partial sequence codes relative to the whole virus genome from1171-1897; Bega (AF049225) E1 and E2 partial sequence codes relative tothe whole-virus genome, from 2253-3490; Trangie (AF049223) E1 and E2partial sequence codes relative to the whole virus genome, from2290-3490; Clover Lane (AF037405, Becher et al., 1998) E1 and E2 partialsequence codes relative to the whole virus genome, from 2360-3510; Bega(AF052303) NS3, NS4a partial sequence codes relative to the whole virusgenome, from 5416-7591; Trangie (AF052304) NS3, NS4a partial sequencecodes relative to the whole virus genome, from 5675-7528.

Extraction of Viral RNA

cDNA was transcribed from Australian BVDV isolates for all of the majorimmunogenic regions (E0, E1/E2 and NS3/NS4A) using standard techniques.Briefly, viral RNA was extracted from infected cells and/or viralpellets using either RNAzol (Biotex Laboratories, Inc) or TRIzol®Reagent (Gibco BRL), according to the manufacturer's instructions. DriedRNA pellets were reconstituted in 10 μl or 20 μl sterile Diethylpyrocarbonate (DEPC) (Sigma) treated water (Sambrook et al., 1989).

Reverse Transcription to Produce cDNA for E1/E2, NS3 and E0 ImmunogenicRegions

cDNA was produced for E1/E2 by reverse transcription by preparing anE1/E2 reverse transcriptase (RT) mixture as described in Table 1. Tubeswere heated in an FTS-960 Thermal Sequencer (Corbett Research) at 37° C.for 50 mins, followed by 70° C. for 10 mins to denature the reversetranscriptase. The RT mix was cooled at 5° C. for 2 mins prior to cDNAamplification by polymerase chain reaction (PCR).

cDNA for NS3 was also prepared by RT by preparing a NS3 RT mixtureaccording to Table 2. Tubes were heated in an FTS-960 Thermal Sequencerat 37° C. for 59 mins, followed by 94° C. for 15 mins to denature thereverse transcriptase prior to cDNA amplification by PCR.

cDNA for E0 was produced by preparing a RT mixture as described in Table3. Tubes were heated in an FTS-960 Thermal Sequencer at 37° C. for 50mins, followed by 70° C. for 10 mins to denature the reversetranscriptase. The RT mix was then cooled at 5° C. for 2 mins prior tocDNA amplification by PCR.

TABLE 1 Reagent Volume Final concentration Supplier X10 PCR buffer 2.0μL X1 Boehringer [100 mM Tris-HCl; 15 [10 mM Tris-HCl; 1.5 Mannheim mMMgCl₂; 500 mM mM MgCl₂; 50 mM KCl; KCl; pH 8.3] pH 8.3] 25 mM MgCl₂ 2.8μL 3.5 mM Sigma molecular biology grade dinucleotide 4 μl 1 mM of eachdNTP Boehringer triphosphate (dNTP) Mannheim containing 5 mM each dATP,dGTP, dCTP and dTTP random hexamers 1 μl 2.5 μM Perkin Elmer (50 μM in10 mM Tris-HCl, pH 8.3; RNase-inhibitor 10 units — Boehringer MannheimM-MLV 12.5 units — Gibco BRL RNA preparation 1 μl — — 20 μl — —

TABLE 2 Reagent Volume Final concentration Supplier X5 first strandbuffer 4 μL X1 (50 mM Tris-HCl, pH Gibco BRL (250 mM Tris-HCl, pH 8.3;55 mM KCl; 3 mM 8.3; 375 mM KCl; 15 MgCl₂:) mM MgCl₂) 0.1 M DTT(dithiothreitol 2 μL 0.01 m Gibco BRL dinucleotide 2 μL 0.5 MmBoehringer triphosphate (dNTP) Mannheim solution (containing 5 mM eachdATP, dGTP, dCTP and dTTP), random hexamers (50 1 μL 2.5 μM Perkin ElmerμM in 10 mM Tris-HCl, pH 8.3) RNase-inhibitor 20 units — (BoehringerMannheim Superscript ™ II (RNase 50 or 100 units — Gibco BRL H⁻ ReverseTranscriptase) RNA preparation 1 μL — — Heated to denature at 65° C. for5 mins and cooled rapidly on ice before use Total volume 20 μL — —

TABLE 3 Reagent Volume Final concentration Supplier X10 PCR buffer 2.0μL X1 Boehringer [100 mM Tris-HCl; 15 [10 mM Tris-HCl; 1.5 Mannheim mMMgCl₂; 500 mM mM MgCl₂; 50 mM KCl; pH 8.3] KCl; pH 8.3] 25 mM MgCl₂ 2.8μL 3.5 mM Sigma molecular biology grade dinucleotide 4 μl 1 mM of eachdNTP Boehringer triphosphate (dNTP) Mannheim containing 5 mM each dATP,dGTP, dCTP and dTTP random hexamers 1 μl 2.5 μM Perkin Elmer (50 μM in10 mM Tris-HCl, pH 8.3; RNase-inhibitor 10 units — Boehringer MannheimM-MLV 12.5 units — Gibco BRL RNA preparation 1 μl — — 20 μl — —

PCR Oligonucleotide Primers

PCR primers for the BVDV isolates, Trangie and Bega, were based onconserved regions of the published sequences for overseas pestivirusisolates. The primers for the BDV isolate, Clover Lane, were made usingits published sequence (Becher et al., 1998). Primers were designedusing the computer programme ‘Primer Designer—Version 2.0’ (Scientificand Educational Software, 1990, 1991) and contained restriction sitesincorporated to enable directional cloning of the cDNA (Tables 4 and 5).

Amplification of cDNA by Polymerase Chain Reaction

The amplification of cDNA from E1/E2, NS3/NS4A and E0 followed a similarprocedure. The amplification reaction was carried out in a total volumeof 100%. To the 20 μl of RT, 8 ml×10 PCR buffer (100 mM Tris-HCl; 15 mMMgCl₂; 500 mM KCl; pH 8.3: Boehringer Mannheim), 7.2 ml 25 mM MgCl₂ (togive a final concentration of 3.3 mM MgCl₂; Sigma, molecular biologygrade), 2.5 units Taq DNA polymerase (Boehringer Mannheim) and 1 μl eachof the sense and antisense primers (30 pmol per ml) were added.

E1/E2 cDNA was initially denatured at 95° C. for 2 mins, followed by 35cycles of denaturation at 95° C. for 30 secs, annealing at 55° C. for 30secs and extension at 72° C. for 1 min. A final extension step of 72° C.for 5 mins was included, before cooling the tubes to 5° C. for 2 mins.

TABLE 4 Location of primer Pestivirus in NADL Protein Primer sequence^(a) sequence ^(h) Trangie 5′ -CGCGGATCCAGTGCTGGCATTTGAAGA- 3′ (SEQ IDNO. 1) 2290 E1/E2^(b*)         Bam HI Bega5′ -CGCGGATCCCAGACTGGTGGCCTTATGA- 3′ (SEQ ID NO. 2) 2253 E1/E2^(c*)        Bam HI CloverLane 5′ -CACGGATCCAGTGCATCAACAACAGCCT- 3′ (SEQ IDNO. 3) 2360 E1/E2^(d*)         Bam HI Trangie5′ -CGCGGATCCAGTTTTGTTTCAAGTTACAATG- 3′ (SEQ ID NO. 4) 1171 EO^(e♦)        BamHI Bega E0^(f♦) 5′ -CGCGGATCCAGTTTTGTTTCAAGTTACAATG- 3′ (SEQID NO. 5) 1171         BamHI Trangie5′ -AACTGCAGACTAGAGTGGTTTGCCAAAGCAACA- 3′ (SEQ ID NO. 6) 5675 NS3/NS4A

        Pst I ^(a) Restriction enzyme sites are shown in bold,^(b)GenBank accession number is AF049223, ^(c)GenBank accession numberis AF04925, ^(d)GenBank accession number is AF037405 Becher et al.(1998), ^(e)GenBank accession number is F049222, ^(f)GenBank accessionnumber is Af049221, ^(g) GenBank accession number is AF052304, ^(h)GenBank accession number is M31182 Collett et al. (1988), ^(*)E1/E2fragments code for a protein containing 69 amino acids (aa) from E1 andfinishing 35 aa before the end of E2, ^(♦)codes for the full length E0protein,

codes for NS3 protein without the serine protease enzyme and includesthe area coding for a T-cell epitope found in CSFV (Pauly et al., 1995).

TABLE 5 Location of primer Pestivirus in NADL Protein Primer sequence^(a b) sequence ^(i) Trangie 5′ -GCGAAGC TTAGGACTCTGCGAAGTAATC- 3′ (SEQID NO. 7) 3490 E1/E2^(c*)        HindIII/Stop Bega 5′ -CATGCCATGGTTAGGACTCTGCGAAGTAATC- 3′ (SEQ ID NO. 8) 3490 E1/E2 ^(d*)        NcoI   Stop CloverLane 5′ -CGCAAGC TTACGCTACCACTGCCAACATGA- 3′(SEQ ID NO. 9) 3510 E1/E2 ^(e*)        HindIII/Stop Trangie 5′ -CGCAAGCTTAGACATCACAGTAAGGGGA- 3′ (SEQ ID NO. 10) 1897 E0 ^(f♦)       HindIIII/Stop Bega E0 ^(g♦) 5′ -CGCAAGC TTAGACATCACAGTAAGGGGA- 3′(SEQ ID NO. 11) 1897        HindIII/ Stop Trangie 5′ -ACGTCCATGGTTAAGCTTGATAGCCTACGTACC- 3′ (SEQ ID NO. 12) 7528 NS3

       NcoI   Stop ^(a) Restriction enzyme sites are shown in bold, ^(b)ln frame stop codon is underlined in the anti-sense primer, ^(c)GenBankaccession number is AF049223, ^(d) GenBank accession number is AF049225, ^(e) GenBank accession number is AF037405 Becher et al. (1998),^(f) GenBank accession number is AF049222, ^(g) GenBank accession numberis AF049221, ^(h) GenBank accession number is AF052304, ^(i) GenBankaccession number is M31182 Collett et al. (1988), ^(*)E1/E2 fragmentscode for a protein containing 69 amino acids (aa) from E1 and finishing35 aa before the end of E2, ^(♦)codes for the full length EQ protein,

codes for NS3 protein without the serine protesase and includes the areacoding for T-cell epitope found in CSFV (Pauly et al., (1995).

The Clover Lane (BDV) PCR mix did not require MgCl₂ and only 1 unit ofTaq DNA polymerase was needed for amplification.

E0 amplification was carried out as described for E1/E2, with theexception that the initial denaturing step was at 94° C. for 2 mins.

Amplification of NS3 cDNA was carried out in a total volume of 50 μlusing the total 20 μl from the reverse transcription reaction, to whichwas added 3 μl×10 PCR buffer (100 mM Tris-HCl; 15 mM MgCl₂; 500 mM KCl;pH 8.3: Boehringer Mannheim), 2 μl 25 mM MgCl₂ (to give a finalconcentration of 3 mM MgCl₂; Sigma, molecular biology grade), 1-2 unitsTaq DNA polymerase (Boehringer Mannheim) and 1 μl each of the sense andantisense primers (25-30 pmol per ml). An initial denaturing step at 94°C. for 3 min was followed by 30 cycles of denaturation at 94° C. for 30sec, annealing at 55° C. for 30 sec and extension at 72° C. for 2 min. Afinal extension step of 72° C. for 5 min was included, before coolingthe tubes to 4° C.

Cloning of PCR Fragments

PCR products were purified with PCR SPINCLEAN™ columns (ProgenIndustries, Limited), according to the manufacturer's instructions. Ifthe PCR reaction produced non-specific bands in addition to the requiredproduct, or subcloning from another plasmid was necessary, the DNA wasfurther purified by elution from a 0.8% agarose gel, using amodification of the method described by Heery (1990).

Purified PCR fragments were digested and ligated into pBlueBacHis A, Bor C baculovirus transfer vectors (MaxBac Baculovirus Expression System,Invitrogen Corporation) containing compatible cohesive overhangs, usingstandard cloning protocols (Sambrook et al., 1989; Current Protocols inMolecular Biology, 1991). A, B or C vectors provide three differentreading frames to achieve protein expression in the baculovirusexpression system (Table 6).

TABLE 6 pBlueBacHis A, B or C transfer Pestivirus Protein vector TrangieE1/E2 C Bega E1/E2 C Clover Lane E1/E2 A Trangie E0 B Bega E0 B TrangieNS3/NS4A B

NS3/NS4A proved difficult to clone directly into the pBlueBacHisbaculovirus transfer vector and was thus first cloned into pCR™IIplasmid (Invitrogen Corporation) using the Invitrogen TA Cloning Kit.The methods for this procedure were carried out according to themanufacturer's instructions. The NS3 fragment was then sub-cloned intothe pBlueBacHis B vector as described for the other fragments of thegenomes.

Transformation of Baculovirus Plasmids with the PCR Fragments

The ligations were transformed into competent E. coli strain Top 10(Invitrogen Corporation), Genotype: F⁻mcrA D(mrr-hsdRMS-mcrBC)f80lacZDM15 DlacX74 deoR recA1 araD139 D(ara-leu)7697 galU galK rpsLendA1 nupG, and/or Sure® E. coli (Stratagene), Genotype: e14⁻(McrA⁻)D(mcrCB-hsdSMR-mrr)_(—)171 endA1 supE44 thi-1 gyrA96 rel A1 lac recB recJsbcc umuc::Tn5 (kan^(r)) uurC[F′ proAB lacI^(a)Z D m15Tn10(Tet^(r))]^(c). Protocols for the preparation of competent cells andtransformation of the bacteria were taken from the Invitrogen MaxBacBaculovirus Expression System Manual Version 1.8.

Screening Bacterial Clones for Plasmid Containing PCR Fragment andPlasmid Purification for Transfection

Bacterial clones containing pBlueBacHis+PCR fragment were identified bygrowing colonies, extracting the plasmids using the boiling miniprepmethod described in Sambrook, et. al. (1989), and then undertakingrestriction digests of the plasmids to verify those containing thecorrect-sized insert. Recombinant plasmids were purified to a levelsuitable for transfection reactions using plasmid purification kits(QIAGEN Pty Ltd., tip-20 or tip-100 columns), according to themanufacturer's instructions.

Production of Purified Recombinant Baculoviruses by Cationic LiposomeTransfection of Sf9 Cells to Produce Recombinant Baculoviruses

Recombinant baculoviruses were produced by co-transfecting linearisedwild-type Autographa californica nuclear polyhedrosis virus (AcMNPV) DNAand baculovirus transfer vector containing PCR fragment into Sf9 cells,by the technique of cationic liposome mediated transfection. This wascarried out according to the Invitrogen MaxBac Baculovirus ExpressionSystem Manual Version 1.8. Some minor modifications were made inrelation to volumes, but these were not significant in terms of theoverall strategy used.

Plague Purifying Recombinant Baculoviruses

Recombinant virus was plaque purified three times before virus masterstocks were prepared, ensuring the virus was cloned from a singleparticle and no wild-type virus was present. Plaque assays were set upaccording the Invitrogen MaxBac Baculovirus Expression System ManualVersion 1.8.

After each round of plaque purification, the recombinant viruses werescreened using a modified Pestivirus antigen-capture ELISA (PACE)(Shannon et al., 1991). The modified method involved supernatant+cells(50 μl/well) being added directly to a blocked, washed ELISA plate, andthe plate incubated for 1 hr at 37° C. Antibody solution (50 μl/well)was then added. The antibody used was either biotinylated goatanti-pestivirus antiserum or individual anti-E2 or anti-NS3 monoclonalantibodies (mAbs). The plate was incubated overnight at 22° C., thendeveloped as described by Shannon et al. (1991), omitting the incubationwith biotinylated anti-mouse IgG for samples that were reacted with thebiotinylated goat antiserum. It should be noted that recombinant,baculovirus-expressed E0 has not been detected in the PACE.

Recombinant Baculovirus Master, Seed and Working Stocks

The master virus stock for each of the recombinant baculovirusesconstructed was made according the Invitrogen MaxBac BaculovirusExpression System Manual Version 1.8. The titre of the stock wasdetermined by a plaque assay, as described above, except that the cellswere overlaid with 1.5% carboxymethylcellulose (CMC, BDH; 6% CMC indeionised water, diluted 1 in 4 with complete TC100+X-gal [125 μg/ml,Boehringer Mannheim]). After 7 days, the blue plaques were counted togive the virus titre.

The seed and working stock were made from the master and seed stock,respectively using a low MOI of 0.1 to 0.5 pfu/ml. All virus stocks werestored at 4° C. for use in vaccine production. For long term storage ofMaster, Seed and Working stocks, each recombinant virus was ampouled andfrozen at −80° C.

Optimisation of Recombinant Protein Production

Sf9 insect-cell suspensions, adapted to Sf-900 II Serum Free Mediaaccording to the protocol described by Gibco BRL (1995), were used tooptimise recombinant protein expression. Two conical flasks, containing50 ml cells (1.5×10⁶ cells per ml), were infected with recombinantbaculovirus at a high and low MOI, between 0.1 and 5.0. A third flaskacted as an uninfected control culture. The 3 flasks were incubated withshaking at 28° C., and 5 ml aliquots removed at 24 hr intervals for upto 7 days.

The samples were centrifuged at room temperature (RT) for 10 min at900×g, and the supernatants carefully removed. The pellets andsupernatants were stored at −20° C. until daily sampling was completed.The amount of specific, recombinant pestivirus protein in the sampleswas then determined using the modified PACE described above. The cellpellets were reconstituted in 200 μl or 250 μl NP-40 (1% [v/v] in PBS),vortexed and centrifuged at RT for 10 min at 900×g. Serial dilutions ofthe pellet extract (in 1% [v/v] NP40) were assayed. The culturesupernatants were assayed undiluted, as well as serially diluted (in 1%[v/v] NP40).

It was found that the cell viability was reduced at the higher rate ofinfection. Therefore, an MOI or 0.1 to 2 was more appropriate. Data forthe optimised expression times and the recombinant protein location inthe insect-cell suspension cultures, either supernatant or pelleted-cellfraction are shown in Table 7.

TABLE 7 Recombinant protein Time to harvest after location in infectedRecombinant protein infection (Hours) cultures Trangie E1/E2 48Supernatant Bega E1/E2 48 Supernatant Clover Lane E1/E2 48 SupernatantBega E0 48^(a) Supernatant + cells^(b) Trangig E0 48^(a) Supernatant +cells^(b) Trangie NS3/NS4A 48 Cells only ^(a)These proteins could not bedetected in the PACE, therefore the time to harvest was based on thetimes at which the other recombinant proteins were harvested. ^(b)Bothsupernatant and cells need to be harvested since the location in theinsect cell suspension cultures could not be accurately determined.

In relation to the expressed E1/E2 proteins, the hydrophobic ‘tail’region of this protein was deliberately omitted when constructing thecDNA of the genome encoding this region and therefore in preparing therecombinant baculoviruses responsible for protein expression in theinsect-cell cultures. This resulted in the majority of these proteinsbeing transported from the insect cells via normal protein-exportpathways and secretion into the cell culture medium. Therefore, maximumprotein recovery was from the supernatant fraction of the insect-cellcultures.

In contrast, the expressed NS3/NS4A protein remains ‘bound’ within theinsect cells and therefore were harvested from pelleted cells at the endof the culture time. Cells were pelleted on a bench centrifuge at 2000×gfor 10 mins, which maximised the harvest of this recombinant protein ina small volume. In the case of the E0 (E^(ms)) recombinant, expressedproteins, the location of these could not be determined and, therefore,culture supernatant plus insect cells were harvested.

EXAMPLE 2 Coupling of Immunogenetic BVDV Proteins to Insect Cell hsps InVitro

The insect cells used throughout the example were Spodoptera frugiperda9 (Sf9) cells (Invitrogen Corporation, USA).

Establishing Sf9 Insect Cells in Monolayers

This method was based on the Invitrogen MaxBac Baculovirus ExpressionSystem Manual, Version 1.8. Frozen cell aliquots (normally 1.0 ml vialsstored in a liquid Nitrogen tank) were thawed rapidly at 37° C., thevial wiped with 70% (v/v) ethanol and the cells transferred to a 75 cm²tissue culture flask containing 15 ml complete TC100 medium (Gibco BRLTC100, supplemented with 10% FCS [Trace Biosciences Pty Ltd] andcontaining penicillin and streptomycin [100 units/ml each]) at roomtemperature (RT).

After the flask had been incubated for 1 hr at 28° C. (ClaysonIncubator, Edwards Instrument Company) to allow the cells to attach, themedium was replaced with 20 ml fresh, complete TC100. The medium waschanged every 48 hrs until the cells were confluent, at which stage theywere passaged as follows. The medium was removed and replaced with 5 mlfresh medium, the cells gently scraped off the bottom of the flask, and1 ml of cell suspension was transferred to a new 75 cm² tissue cultureflask containing 19 ml of complete TC100 at RT. After rocking the flaskto distribute the cells evenly across the bottom, the flask was placedat 28° C. and the cells grown as described above.

Culture of Sf9 Insect Cells in Suspension

This method was based on the Invitrogen MaxBac Baculovirus ExpressionSystem Manual, Version 1.8. Cells were grown as suspension culturesafter they had been passaged 3-4 times as a monolayer culture. Themedium used for these cultures was complete TC100 containing 1% (v/v)pluronic F-68 (10% solution; Gibco BRL). To produce the suspensioncultures, medium was first removed from the cells which were a confluentmonolayer and immediately replaced with 5 ml of fresh medium. The cellswere gently scraped from the bottom of the flask and the cell suspensiontransferred to a 100 ml, screw-topped, conical flask containing 25 ml ofmedium containing pluronic. The flask was incubated at 28° C., shakingon an orbital shaker (Bio-Line; Edwards Instrument Company) for at least1 hr at 80 rpm before increasing the shaking speed to 110 rpm for theremainder of the growth period. When the cell density had reachedapproximately 2×10⁶ cells/ml, an additional 30 ml of medium was added.Cells were then grown on in 50 ml volumes and passaged when the celldensity reached approximately 2×10⁶ cells/ml. At this time, cells werediluted in medium to give approximately 0.5×10⁶ cells/ml, and incubatedat 28° C., shaking at 110 rpm, until passaged again.

Adaptation of Sf9 Insect Cells to Grow in Suspension Culture inSerum-Free Medium

Insect cells were adapted to grow in suspension culture in serum-freemedium according to the protocol described by Gibco BRL (1995). Cellsthat had been growing in complete TC100 with pluronic were transferredto 50 ml of serum-free medium (Sf-900 II SFM; Gibco BRL) containingpenicillin and streptomycin (100 units/ml each) at a density of 5×10⁵cells/ml. The cells were grown on to a density of 2×10⁶ cells/ml, thenpassaged at a density of 5×10⁵ cells/ml. This was continued until a celldensity of 2×10⁶ cells/ml, with a cell viability of at least 80%, wasreached. The cells were then considered to be adapted to grow inserum-free medium, and subsequently were passaged using serum-freemedium in the same way as described for cells grown in complete TC100medium with pluronic F-68, as described for the culturing of Sf9 insectcells in suspension.

Requirements for Sf9 Cells Used to Express Recombinant PestivirusProteins

Sf9 cells in serum-free medium, used for the expression of recombinantpestivirus proteins, were required to be below 30 passages in culture.Conical flasks (500 ml) were seeded with Sf9 cells at a density 0.5×10⁶cells/ml in a final volume of 150 ml. When the cells in these flasksreached a density of 1.0-1.5×10⁶ cells/ml, they were infected with theappropriate recombinant baculovirus encoding the required, expressedpestivirus protein.

Production of the Recombinant Baculoviruses Encoding Pestivirus Proteins

Production of recombinant baculoviruses and expression of recombinantpestivirus proteins is described in Example 1.

Multiplicity of Infection

The Sf9 insect cells were infected at a multiplicity of infection (MOI)ranging between 0.1 and 2.0, depending on the individualrecombinant-baculovirus stock titre. Results are shown in Table 8.

Induction of Heat Shock Proteins in Cultures of Sf9 Cells Infected withRecombinant Baculoviruses

The experimental times and temperatures used to induce the production ofheat shock proteins in insect-cell cultures were optimised for Sf9insect-cell cultures expressing specific pestivirus recombinantproteins, as set out below.

Heat Shock Conditions for NS3/NS4A Recombinant Baculovirus-InfectedCultures

Sf9 insect cells infected with the recombinant baculovirus expressingthe truncated pestivirus NS3/NS4A protein were incubated at 28° C., withshaking at 110 rpm, for 48 hours. The infected cell cultures flasks werethen placed in a 43° C. water bath along with a similar ‘dummy’ cellculture flask containing a thermometer in 150 ml of water. A 10 minincubation was started when the thermometer reached 43° C., and thenevery 2 mins, the flasks were given a gentle mix by swirling the mediumand cells within the flask. This was determined to be the optimalheat-shock conditions for Sf9 cells expressing pestivirus recombinantproteins. The cell culture flasks were then placed back into theincubator (at 28° C.), with shaking at 110 rpm, for a further 2-hrperiod to allow the insect-cell, heat-shock proteins (hsps) to beexpressed and coupled to the pestivirus NS3 recombinant protein.

TABLE 8 Recombinant EMAI Virus Titre Multiplicity Baculovirus numberStock (pfu/ml) of Infection AcMNPV + Z044 Master 2.8 × 10⁷ 1.0 TrangieE1/E2 AcMNPV + Z376 Seed 1.0 × 10⁷ 1.0 Bega E1/E2 AcMNPV + Z361 Working4.35 × 10⁷  2.0 Clover lane E1/E2 AcMNPV + Z341 Master 3.2 × 10⁶ 0.2Bega E0 AcMNPV + Z293 Seed 2.2 × 10⁷ 1.0 Trangie E0 AcMNPV + Z346 Master2.0 × 10⁶ 0.2 Trangie NS3/NS4A

Heat Shock Conditions for E1/E2 and E0 Recombinant Baculovirus-InfectedCultures

Sf9 insect cells infected with recombinant baculoviruses expressingeither E1/E2 or E0 pestivirus proteins were incubated at 28° C., withshaking at 110 rpm, for a period of 24 hr. The infected cell cultureswere then heat shocked exactly as described above (10 min at 43° C.).The cell cultures in their flasks were then returned to the incubator(28° C.) and the cells cultured, with shaking at 110 rpm, for a furtherperiod of 24 hr. In this system, the cultures expressing the E1/E2proteins were heat shocked at 24 hr, as opposed to 48 hr for theNS3/NS4A protein, to ensure that the E1/E2 recombinant proteins werecoupled to insect cell hsps prior to their transport out of the Sf9cells and into the cell culture medium.

Since it had not yet been determined whether the recombinant E0pestivirus proteins were secreted from insect cells, or remained withinthe cells themselves, Sf9 cultures producing the E0 pestivirus proteinswere also heat shocked at 24 hr after infection with the recombinantbaculoviruses.

Heat Shock Conditions for Uninfected, Control-Cell Cultures

Uninfected Sf9 insect cell cultures were incubated at 28° C. (shaking at110 rpm) for a period of 48 hr. These control cell cultures were heatshocked exactly as described above. The cell culture flasks were thenreturned to 28° C. in the incubator, with shaking at 110 rpm, for afurther 2 hr incubation period to allow the insect cell heat shockproteins to be formed by the stressed cells.

Harvesting of Individual Recombinant Pestivirus Proteins from Sf9 CellCultures [NS3 Recombinant Protein+Heat-Shock Proteins (hsps)]

Cells were separated from the medium by centrifugation at 2000×g for 10mins. The cell pellet, containing the NS3/NS4A antigen, was thenresuspended in one sixth of the original volume using serum-free medium(Sf-900 II SFM; Gibco BRL) containing leupeptin (protease inhibitor, ICNBiomedicals, Inc) at a concentration of 5 μg/ml. This gave an effectivesix-fold concentration of the cells plus recombinant NS3/NS4A antigen.The cells were then freeze/thawed twice at −80° C. to break downcellular membranes and release the NS3 recombinant protein, togetherwith recombinant baculovirus, into the medium.

Harvesting of Individual Recombinant Pestivirus Proteins from Sf9 CellCultures [E1/E2 Recombinant Protein+Heat-Shock Proteins (hsps)]

Cells were removed from the medium by centrifugation at 2000×g for 10mins. Leupeptin (protease inhibitor) was again added to the supernatant,containing the expressed E1/E2 protein, to give a final concentration of5 μg/ml. The addition of the protease inhibitor prevented degradation ofthe expressed proteins.

Harvesting of Individual Recombinant Pestivirus Proteins from Sf9 CellCultures [E0 Recombinant Protein+Heat-Shock Proteins (hsps)]

In the case of insect-cell cultures expressing this particular protein,the whole culture (cells plus medium) was harvested and leupeptin addedto give a final concentration of 5 μg/ml. The culture was thenfreeze/thawed twice to break down cellular membranes, releasing both therecombinant baculoviruses and any cell-associated E0 proteins into themedium.

Harvesting of Individual Recombinant Pestivirus Proteins from Sf9 CellCultures [Control Cells+Heat-Shock Proteins (hsps)]

Control (uninfected) cultures were harvested as described above forNS3/NS4A However, the control cells were concentrated 10-fold.

Beta-Propiolactone (βPL) Inactivation of Recombinant Baculoviruses

βPL inactivation (using β-propiolactone, Sigma Aldrich Fine Chemicals)was carried out twice on all recombinant protein preparations producedby the baculovirus-vector expression-vector system, and for thecontrol-cell preparation. The standard method employed by theCommonwealth Serum Laboratories (CSL, “Inactivation of Baculovirus usingBeta-Propiolactone, 1998”) was used in all cases. To ensure no residualinfectious baculovirus was left in the “inactivated” material, eachpreparation was passaged three times in Sf9 monolayers. The final passwas titrated in an Sf9 plaque assay, using 1.5% carboxymethylcellulose(CMC, BDH; 6% CMC in deionised water diluted, 1 in 4 with completeTC100) containing 125 mg/ml X-gal (Boehringer Mannheim) as the overlay.Plaque assays were set up according the Invitrogen MaxBac BaculovirusExpression System Manual, Version 1.8, and the plaque assay read on day7. There was no evidence of live, infectious baculovirus present in anyof the preparations used to formulate the subunit vaccine, thus meetingthe Australian Quarantine Inspection Service (AQIS) requirements for theuse of the experimental vaccine in food-producing animals.

Concentration of the Recombinant E0 and E1/E2 Protein Preparations.

The Sf9 cells expressing the E0 recombinant proteins were separated fromthe medium after inactivation (as above) by centrifugation at 2000×g for10 min and these cells were then stored at 4° C. pending their use. Thesupernatants containing recombinant E0 proteins were then concentratedfive times in separate Amicon Ultrafiltration Cell steps, according tothe manufacturer's instructions. The concentrated E0 protein-containingsupernatant was then re-mixed with the E0 Sf9 cells to prepare thefinal, concentrated preparation.

In the case of the inactivated E1/E2 recombinant proteins, thesupernatant fractions only were concentrated using the AmiconUltrafiltration Cell. These proteins are all secreted from therecombinant-baculovirus infected cells and therefore the cell fractionis discarded.

Determination of the Amount of Recombinant Pestivirus Protein in EachPreparation by Titration in the Pestivirus Antigen Capture ELISA (PACE).

The amount of recombinant protein, after βPL inactivation, was assayedby titrating each individual recombinant protein preparation in themodified PACE (see Shannon et al., 1991). The modification of thepublished method involved sample (50 μl/well) being added directly to ablocked, washed ELISA plate, and the plate incubated for 1 hr at 37° C.Antibody solution (50 μl/well) was then added. The antibody used waseither biotinylated goat anti-pestivirus antiserum (pAb) or individualanti-E2 or anti-NS3 monoclonal antibodies (mAbs). The plate wasincubated overnight at 22° C., then developed as described in Shannon etal. (1991), omitting the incubation with biotinylated anti-mouse IgG forsamples that were reacted with the biotinylated goat antiserum.

It should be noted that recombinant E0 protein is not able to bedetected in this assay system since the protein failed to react witheither of the polyclonal or monoclonal antibodies. Therefore, it wasassumed that this protein was similar in concentration to thosedetermined for the analogous E1/E2 expressed structural glycoproteins.

Summary of the Recombinant Proteins Incorporated in the Subunit Vaccine

The recombinant pestivirus proteins Trangie NS3/NS4A, Trangie E0, BegaE0, Trangie E1/E2, Bega E1/E2, Clover Lane E1/E2, together with theControl cells, were prepared by the methods described in this example.However, Bega and Trangie E1/E2 were not heat shocked, Trangie E1/E2 wasconcentrated six times instead of five and the BDV Clover Lane. E1/E2recombinant protein was processed as described for the recombinant E0protein preparations.

Recombinant, Experimental Subunit Vaccine

The composition of the vaccine preparations used in Example 4 are setout in Table 9. In summary, each dose of the recombinant pestivirusvaccine contained: 1 ml Bega E0, 1 ml of Trangie E0, 1 ml of Clover LaneE1/E2, 0.5 ml Trangie E1/E2, 1 ml of Bega E1/E2 and 0.3 ml of TrangieNS3/NS4A. Thimerosal (mercuric compound, Sigma Aldrich) was added to thevaccine mixture to help prevent bacterial contamination, with a finalconcentration in the vaccine of 0.1% (w/v). Isocomatrix adjuvant(Commonwealth Serum Laboratories, Australia) was used at the rate of 2mg incorporated in each vaccine dose. The formulated vaccine was storedat 4° C. until injected into the cattle (initial dose followed by a secdose 4 weeks later).

Control Vaccine

The formulation of the Control vaccine is also set out in Table 9. Insummary, each dose of control vaccine contained 4.8 ml of thecontrol-cell preparation. Thimerosal was again added to the controlvaccine to help prevent bacterial contamination, the final concentrationin the vaccine being 0.1% (w/v). Isocomatrix adjuvant (CSL) wasincorporated at 2 mg per vaccine dose, in line with the rate used in theexperimental vaccine. The control vaccine preparation was stored at 4°C. until required. Two doses were given to the control animals in thetrial on the same days as the experimental subunit vaccine wasadministered to the vaccinated animals.

TABLE 9 Summary of Subunit, expressed - protein Vaccine preparationTrangie NS3/NS4A Labelled: TNS3 8 Apr. 1998 MOI 0.2, grown for 48 hrs inSFM Heat shocked at 43° C. for 10 mins Placed back in incubator for 2hrs with shaking ONLY cells harvested (S/N discarded), therefore [ ] 6×Leupeptin added to give 5 ug/ml Freeze/thawed 2 times BPL inactivated 2times using CSL standard method Stored at −80° C. (Block 5) Trangie E0Labelled: TE0 23 Apr. 1998 MOI 1, grown for 48 hrs in SFM Heat shockedat 43° C. for 10 mins Placed back in incubator for 24 hrs with shakingCells + S/N harvested Leupeptin added to give 5 ug/ml Freeze/thawed 2×BPL inactivated 2 times using CSL standard method AMICON concn. 5×Stored at −80° C. (block 5) Bega E0 Labelled: BE0 23 Apr. 1998 MOI 0.2,grown for 48 hrs in SFM Heat shocked at 43° C. for 10 mins Placed backin incubator for 24 hrs with shaking Cells + S/N harvested Leupeptinadded to give 5 ug/ml Freeze/thawed 2× BPL inactivated 2 times using CSLstandard method AMICON concn 5× Stored at −80° C. (Block 5) Clover LaneE1/E2 Labelled: CLE2 23 Apr. 1998 MOI 2, grown for 48 hrs in SFM Heatshocked at 43° C. for 10 mins Placed back in incubator for 24 hrs withshaking Cells + S/N harvested Leupeptin added to give 5 ug/mlFreeze/thawed 2 times BPL inactivated 2 times using CSL standard methodAMICON concn. 5× Stored at −80° C. (Block 5) Trangie E1/E2 Labelled: TE28 May 1998 MOI 1, grown for 48 hrs in SFM ONLY S/N harvested Leupeptinadded to give 5 ug/ml Freeze/thawed 2 times BPL inactivated 2 timesusing CSL's standard method AMICON concn. 6× Stored at −80° C. (Block 5)Bega E1/E2 Labelled: BE2 8 May 1998 MOI 1, grown for 48 hrs in SFM ONLYS/N harvested Leupeptin added to give 5 ug/ml Freeze/thawed 2 times BPLinactivated 2 times using CSL standard method AMICON concn. 5× Stored at−80° C. (Block 5) Control Vaccine Labelled: SFM-Sf9 24 Jun. 1998 Grownfor 48 hrs in SFM Heat shocked at 43° C. for 10 mins Placed back inincubator for 24 hrs with shaking Cells harvested and taken up in 50 mlSFM therefore [ ]10× Leupeptin added to give 5 ug/ml Freeze/thawed 2times BPL inactivated once using CSL standard method Stored at −80° C.(Block 5)

Mixing of Recombinant Proteins to Produce the Subunit Vaccine

Rec Antigen 1× DOSE 27× DOSES BEO 1 ml 27 ml TE0 1 ml 27 ml CLE1/E2 1 ml27 ml TE1/E2 0.5 ml 13.5 ml TNS3/NS4A 0.3 ml 8.1 ml BE1/E2 1 ml 27 mlTotal Volume 129.6 ml

Added 1.29 ml Thimerosal to 129.6 ml vaccine mix, took out 9.6 ml andplaced into 1 ml aliquots for storage at −20° C. (freezer in Block 5 eggroom).

To the remaining 120 ml, added 32.5 ml Iscomatrix adjuvant and stirredfor 2 mins to mix well.

Aliquoted into 2 containers i.e 68 ml/container and stored at 4° C.

Set up vaccine in 10 ml syringes with 18 gauge needles—6 ml/dose finalvolume.

Control Vaccine

1 dose 6 doses Control cell preparation 4.8 ml 28.8 ml Iscomatrixadjuvant 1.3 ml 7.8 ml Thimerosal 0.048 ml 0.29 ml

The control vaccine was set up in 10 ml syringes with 18 gauge needles—6ml/dose final volume

EXAMPLE 3 The Effect of the Subunit Vaccine on Australian Cattle Formatof the Subunit Foetal Protection Trial

A total of 22 pestivirus antibody negative, non-pregnant heifers wereselected for the trial. A group of animals (n=10) were vaccinated twice,4 weeks apart, with the subunit protein vaccine (6 ml) as prepared inexamples 1 and 2. A further group of animals (n=12) were vaccinated withthe control preparation (6 ml). All animals were bled at regularintervals and the concentrations of both anti-E2 and anti-NS3 antibodieswere determined using the complex-trapping-blocking ELISA (CTB-ELISA)format as carried out by the Elizabeth Macarthur Agricultural Institute(EMAI).

Immediately after the second vaccination, the animals were synchronisedfor oestrus. Insemination occurred immediately after oestrus wasdetected. All animals were judged to have become pregnant and havedeveloping foetuses of greater than 6 weeks of age, at 11 weeks afterthe second vaccination, a time considered to be the most susceptible forinfection with the challenged BVDV isolate. The heifers were thenchallenged with a dose (3×10⁶ TCID₅₀) of the live heterologous BVDVisolate Glen Innes.

Six weeks following viral challenge, all heifers were slaughtered at anexport abattoir (Mudgee) in the two groups. The foetuses were collectedfrom pregnant heifers. That is, there were 7 foetuses from the 10animals in the vaccinated group and there were 9 foetuses from the 12animals in the control group.

Individual foetal tissues were collected under sterile conditions.Several methods were employed to test for the presence of BVDVinfection. Firstly, two antigen-capture ELISAs specific for either E2antigens or NS3 antigens were used. Secondly, a panel of monoclonalantibodies was used to detect infected cells isolated using standardtechniques and immunoperoxidase (IPX) staining. Thirdly, a 5′-UTRvirus-specific RT-PCR was used. The combination of these methods gave asensitive and specific detection of infected versus non-infectedfoetuses collected from the heifers.

Effect of E2 Subunit Vaccine on Cattle Immune Response to BVDV

The average concentration of anti-E2 (neutralising) antibody in both thevaccinated and control groups of heifers, before and after vaccination,and after live virus challenge, is shown in FIG. 1.

The average concentration of anti-E2 antibody plotted over timeindicated that the subunit vaccine resulted in very high concentrationsof anti-E2 antibody in the vaccinated group. High titers of antibodycommenced as early as 2 weeks after the administration of the seconddose of vaccine. The concentration of anti-E2 antibody in vaccinatedheifers was significantly higher than in the control group. Theconcentration of anti-E2 in the vaccinated group declined slightly overthe preceding 9 weeks, but still remained significantly higher than thecontrol group of heifers.

A rapid anamnestic rise in the concentration of E2 antibody in thevaccinated group was observed at 7 days post challenge with the live BVDvirus, which continued to rise until 9 days post challenge, where itremained at a sustainable maximum concentration. In contrast to thistrend, an increase in the concentration of anti-E2 antibody was onlyobserved in the control group after challenge with the live virus. Theonset of a normal response in the control group was then observed, withthe average concentration of anti-E2 antibody beginning to develop at 14days post challenge. However, a maximum response was not reached until 3to 4 weeks post challenge.

Thus the vaccination of pregnant heifers with the subunit vaccinecreates an immune response in the heifer during the first 4 to 7 daysafter viral infection. This is an important stage during which the livevirus crosses the placenta to the developing fetus. These resultsclearly indicate that the replication of the live virus was antagonisedin the subunit vaccinated group of heifers (n=10). This is the firsttime that such a response has been reported for a subunit vaccine.

Effect of NS3 Subunit Vaccine on Cattle Immune Response to BVDV

The concentration of anti-NS3 antibody in both vaccinated and controlgroups of heifers over time is shown in FIG. 2. Surprisingly, there wasno anti-NS3 antibody detected in the vaccinated heifers aftervaccination. The reason for this is not yet known. However, this resulthas a great potential for the development of a “marker” vaccine. All“naturally infected” animals develop anti-NS3 antibodies 21 days afterinfection with BVDV. Since animals vaccinated with the subunit vaccinedo not develop anti-NS3 antibodies (discussed below), they are easilydistinguishable from “naturally infected” animals.

It is likely that anti-NS3 protein results in the generation of a strongcell-mediated immune response through the induction of CD8+ cytotoxic Tcells although failing to elicit an antibody response.

After challenge with the live virus, vaccinated heifers (7 out of 10)showed no significant development of anti-NS3 antibodies until 5 to 6weeks post challenge. The remaining three vaccinated heifers developedanti-NS3 antibodies 3 to 6 weeks post challenge. However, theconcentration of antibodies was significantly lower than the controlgroup of heifers. In contrast, the control heifers (n=12) developed anormal antibody response commencing 14 to 18 days post challenge,reaching a peak 4 weeks post challenge (FIG. 2).

These results clearly indicate that the replication of the live viruswas inhibited in the subunit vaccinated group of heifers (n=10). This isthe first time that such a response has been reported for a subunitvaccine. It is evident that the postulated early onset of CTL responsesdirected against infected cells prevented the replication of the virus.Thus there was insufficient virus circulating in the vaccinated animalsto cross the placenta and infect the fetus.

The Concentration of Neutralising Antibodies Induced by the SubunitVaccine

Serum neutralisation tests (SNTs) were carried out using different BVDVisolates to investigate the concentration of neutralising antibodiesinduced by the subunit vaccine, and to determine the anamnesticresponses resulting from live virus challenge.

The results of this experiment are shown in Table 10. As expected fromthe results shown in FIG. 1, no antibody response was observed prior tovaccination in the vaccinated group of heifers. However, after thesecond vaccination, a very good anti-E2 neutralising antibody response(average titre of 1 in 1000) was observed in the 2 BVDV isolatesassociated with the vaccine (Trangie and Bega). In contrast, there was avery low response of neutralising antibody (average titre of 1 in 50)against the sheep BDV isolate (Clover Lane) even though the recombinantE2 protein from this virus was incorporated in the vaccine incombination with hsps. However, the resulting concentration ofneutralising antibody was greater from this subunit vaccine than thatachieved with the use of inactivated whole Clover Lane virus in aprevious experiment (results not shown).

SNTs conducted on the heterologous challenge virus Glen Innes indicateda surprisingly high cross-reactivity (average titre 1 in 1200) at 4weeks after the second dose of vaccine. This finding confirms that thecombination of E2 proteins results in good cross protection againstheterologous viruses. An even more distant BVD virus Braidwood showed alower concentration of neutralising antibodies (average titre of 1 in400) but does correspond with a significant antibody production againstinfection with this virus.

The SNT assays carried out on serum collected from the vaccinatedanimals at 7 and 14 days post challenge (Table 10) were even moresurprising. Assays were conducted using each of the 3 virusesrepresented in the subunit vaccine. It is evident from the results thatthere was an extremely high anamnestic response in the anti-E2 antibodylevels at just 7 days post challenge. Average SNT for both Trangie andBega BVDV viruses were in the order of 1 in 14 000 to 16 000 at 7 daysbut rose to an extraordinary concentration by day 14 post challenge. At14 days post challenge, the average titre against Trangie was 1 in 180000, with 2 animals having titres as high as 1 in 512 000. Similarly,titres against Bega were on average 1 in 100 000 at 14 days, with 3animals having titres against Bega of in 256 000. The magnitude of thesetitres is rarely seen in “naturally infected” animals.

TABLE 10 SNT at 4 weeks post second vaccination with SNT at 7 days postchallenge SNT at 14 days post challenge subunit vaccine

with Glen Innes virus

with Glen Innes virus

Vaccinated Clover Glen Braid- Clover Clover Animal Number Trangie BegaLane Innes wood Trangie Bega Lane Trangie Bega Lane Q239 >2056 >205632 >2056 1024 12800 12800 800 12800 64000 8000 Q269 512 128 16 256 643200 3200 ~4 16000 16000 64 Q300 256 512 128 512 128 6400 12800 80064000 32000 8000 Q306 1028 256 16 1028 256 12800 51200 1600 64000 640004000 Q324 1028 512 128 1028 256 3200 1600 400 64000 16000 4000 Q354 12864 <4 128 16 3200 1600 100 64000 64000 2000 Q355 1028 >2056 32 2056 51212800 12800 1600 512000 256000 16000 Q379 2056 >2056 32 >2056 1024 2560012800 1600 128000 64000 8000 Q382 512 512 64 >2056 64 6400 6400 100256000 256000 8000 R346 >2056 >2056 64 >2056 1024 51200 51200 3200512000 256000 64000 Mean Titre 1028 1020 50 1200 436 13700 16600 102418000 108000 12250 (Range) (128- (64-> (0- (128-> (16- (3200- (1600-(~4- (16000- (16000- (64- 2056) 2056) 128) 2056) 1024) 51200) 51200)3200) 512000) 256000) 64000)

= scrum dilutions started at 1 in 4; 2-fold dilutions to end-point;

= scrum dilutions started at 1 in 1000 for Trangie and Bega, 1 in 100for Clover Lane; 2-fold dilutions to end-point.

TABLE 11 Reciprocal of SNT (NPLA) Titre (4 weeks after secondvaccination in both trials) Inactivated Vaccine Vacci- 1112/96 SubunitVaccine 584/98 nated (T + B + CL) (T + B + CL) Animal Glen Clover GlenClover Number Trangie

Innes

Lane

Trangie

Innes

Lane

1 1024 512 10 >2048 >2048 32 2 256 512 20 512 256 16 3 256 512 20 256512 128 4 40 64 10 1024 1024 16 5 50 50 10 1024 1024 128 6 200 256 8 128256 <4 (0) 7 — — — 1024 2048 32 8 — — — 2048 >2048 32 9 — — — 512 >204864 10 — — — >2048 >2048 64 Mean 300 300 12 1028 1200 50 Titre (Range)(40- (50- (8- (128- (128-> (0- 1024) 512) 20) 2048) 2048) 128)

Comparison of the neutralising titres against 3 viruses measured incattle at 4 weeks after vaccination with either the inactivated,whole-virus vaccine or the non-infections, subunit vaccine containingrecombinant proteins. (

) Trangie (BVDV isolate) and Clover Lane (BDV isolate) incorporated inboth vaccines. In subunit vaccine, Clover Lane E2 recombinant proteinwas coupled in vivo with heat shock proteins (hsps); (

)=Glenn Innes was the challenge live virus used in both trials (a BVDVisolate clearly distinct from the vaccine viruses).

Thus it can be concluded that the subunit vaccine had a significanteffect in “priming” animals for a reaction against live virus challengethat has not previously been observed.

The SNT assays indicated a strong “priming” response against the BDVisolate Clover Lane (Table 10). At 7 days post challenge with a totallyunrelated live virus (BVDV Glen Innes) the SNTs against Clover Laneindicated a titre of 1 in 1024 (increased from 1 in 50 observedfollowing vaccination). The titre against Clover Lane rose at 14 days toan average of 1 in 12 000, with one animal giving a titre of 1 in 640000 against the sheep isolate. This provides further evidence that thesubunit vaccine provides wide spread protection against all Australiancattle and sheep pestiviruses. This protection is far greater thanpresently available with inactivated whole virus vaccines.

Additional SNTs were carried out against 3 Australian pestiviruses. Twogroups of animals were vaccinated with two different vaccines derivedfrom the Trangie+Bega+Clover Lane isolates and were thus directlycomparable. In the first group, animals were vaccinated with anexperimental inactivated whole virus vaccine. The second group ofanimals was vaccinated with the subunit vaccine (+hsps). Serum wascollected from the vaccinated cattle in both groups 4 weeks after 2doses of the vaccine.

The results for the SNTs against the 3 viruses are shown in Table 11. Acomparison of all 3 viruses showed that the subunit vaccine resulted intitres at least 4 times higher than the corresponding titres induced bythe inactivated vaccine. In addition, cross neutralisation occurred forboth vaccines against the totally unrelated BVDV isolate “Braidwood”,which showed a similar 4 fold increase in the titre at 4 weeks postvaccination with the subunit vaccine when compared to the inactivatedvaccine (results not shown).

Thus the requirement for a wider ranging vaccine in all cattle-producingcountries is met by the development of this subunit vaccine.

Effect of Subunit Vaccines on Transfer of BVDV to the Fetus

Tissue samples collected from foetuses (n=7) obtained from the pregnantvaccinated heifers and from foetuses (n=9) obtained from the pregnantcontrol heifers were tested at EMAI using 3 different BVDV-specificassays (Table 12) as described previously. It was apparent from all 3tests that there was no BVDV infections in any of the 7 foetusesobtained from the pregnant vaccinated heifers. In contrast, 5 of the 9foetuses in the control group were infected as shown by the virusisolation and RT-PCR assays.

Therefore, it was concluded that vaccination gave 100% protectionagainst a live, heterologous BVDV challenge at a time when there is amaximum chance of transferring virus into the developing foetus.

TABLE 12 Virus Isolation & 5′ UTR RT-PCR Foetus Antigen ELISA (PACE)Results Results harvested (S/N Ratios)

RT- from E2 + VI PCR Animal No. E2

NS3 NS3 Final Result 2nd Pass (+/−) Vaccinates Q239 1.0 1.1 −ve −ve −ve−ve Q306 1.0 0.9 −ve −ve −ve −ve Q324 1.0 1.2 −ve −ve −ve −ve Q354 1.01.1 −ve −ve −ve −ve Q355 0.9 1.0 −ve −ve −ve −ve Q379 1.2 1.1 −ve −ve−ve −ve R346 1.0 0.9 −ve −ve −ve −ve Controls Q240 1.2 1.1. 1.0 −ve −ve−ve Q316

0.7 0.6 <1.0 −ve +ve

+ve Q352 1.0 1.0 1.0 −ve −ve −ve Q373 0.9 1.1 1.0 −ve −ve −ve Q388 2.913.6 15.8 +ve +ve +ve R298 4.9 10.8 11.1 +ve +ve +ve Q337 1.0 0.9 1.0−ve −ve −ve Q372 3.2 12.8 14.1 +ve +ve +ve Q385 2.3 16.2 17.6 +ve +ve+ve

= signal-to-noise ratios. Ratio >2.0 are positive in PACE on foetaltissues.

= Positive E2 results low. Results confirmed by high S/N ratios with NS3monoclonals on positive tissues.

= foetus was clearly dead in utero. Results confirmed foetus wasinfected.

Weak virus isolation positive-only individual cells strained onmicroplate. Virus titre therefore low in this dead fetus. Resultsconfirmed by diagnostic RT-PCR

EXAMPLE 4 The Effect of the Subunit Vaccine on Australian Sheep

Two additional trials on subunit proteins were conducted in sheep toinvestigate further the protective effect of these vaccines. Thesetrials were conducted to examine the effect of a combination of proteins(coupled with heat-shock proteins) compared to a single viral proteinwith/without heat shock proteins in affording foetal protection againstthe transfer of the same live, heterologous pestivirus used in thecattle trial. Although the same BVDV isolate (‘Glen Innes’) was used forchallenge in the sheep, the dose was reduced to 2×10⁵ TCID₅₀ per sheep,or 50 times less live virus than was used for challenge in cattle.

In the first of these trials involving 24 sheep, the same cattle subunitvaccine was prepared using two different commercial adjuvantpreparations. Two groups of 8 sheep were each given 2 doses of subunitproteins while 8 sheep were injected with a control preparationcontaining insect cells, but no pestivirus proteins. Antibody assays foranti-E2 and anti-NS3 antibodies in the serum of vaccinated and controlsheep over the course of the study were carried out in the same way asfor the cattle subunit trial. A ram was put in with the ewes immediatelyafter the second vaccination. At 10 weeks after vaccination, when all ofthe ewes were pregnant, they were challenged with live, heterologouspestivirus (Glen Innes virus). Five weeks later, all of the ewes wereslaughtered and the foetuses collected for assays to establish whetherthey showed pestivirus infection in the foetal tissues. The absence ofany detectable pestiviruses in the foetal tissues showed that there hadbeen no transfer of virus from the ewe to her foetus. This equated withcomplete protection and was measured only in the two vaccinated groupsof animals (see below).

The antibody responses in the vaccinated and control groups of sheep inare presented in FIGS. 3 and 4. It can be seen that the vaccinatedanimals responded by producing high levels of anti-E2 antibody(equivalent to ‘neutralising antibody), beginning 2 weeks after thesecond dose of vaccine. This was a similar response to the anti-E2antibody response in cattle vaccinated with the same subunit vaccine.However, it is noteworthy that the absolute levels of anti-E2 antibodymeasured by ELISA in the vaccinated sheep (FIG. 3) were less than thecomparable levels measured in vaccinated cattle. Like cattle, there wasan immediate, anamnestic response in the vaccinated sheep with highlevels of anti-E2 antibody measured at just 7 days after challenge withthe live virus. This is good evidence that the subunit-vaccinated sheepproduced early, protective, antibody responses, just as vaccinatedcattle showed in Example 3.

As in cattle, sheep did not produce any anti-NS3 antibody followingvaccination (FIG. 4) confirming that the subunit preparation is a‘marker vaccine’. Following live-virus challenge of the vaccinatedsheep, it is highly significant that 14 of the 16 sheep had not producedany anti-NS3 antibody by 5 weeks after challenge, when the ewes wereslaughtered. The remaining 2 sheep had produced only moderate anti-NS3antibody levels, when compared to the levels of anti-NS3 antibody in thecontrol, unprotected sheep at the same time point (see FIG. 4). Takentogether, these results are clear evidence that subunit vaccination inthe sheep reduced viral replication in the ewes after challenge with thelive BVD virus. Thus, the anti-NS3 antibody results in sheep confirm andextend the results seen in subunit-vaccinated cattle. It was thereforeof importance to see if the reduced viral replication in the sheepequated with protection from transfer of virus into the foetuses of thevaccinated ewes.

The foetal assay results showed that there was no difference in thefoetal protective index between the 2 vaccinated groups. There wastherefore no effect of changing the adjuvant used in the 2 formulationsof vaccine. The results from both groups were able to be pooled, giving16 foetuses from the vaccinated ewes for comparison with 8 foetuses fromthe control, unprotected ewes. Analyses of all results showed that,overall, there was 56% complete foetal protection ( 9/16 foetusesprotected) in the vaccinated ewes compared to 100% infection rate ( 8/8foetuses infected) in the control ewes. However, the rate of transfer oflive pestivirus into 5/7 of the positive foetuses collected from thevaccinated ewes was dramatically reduced compared to the foetuses fromthe control ewes (Table 13). This meant that only 2/16 foetuses in thevaccinated ewes contained similar amounts of virus to the levels in thecontrol-group foetuses. Thus, in 87% of the vaccinated ewes, there waseither no transfer of virus, or a severely-restricted transfer.

This shows that the subunit vaccine, although not as effective in sheepas it is in cattle, still gives a high level of protection in vaccinatedewes. Both trials showed significant, strong protective results for thesubunit vaccine preparation. As discussed below, the difference inresults between cattle and sheep is likely to be the result of lessresistance in sheep to the effects of a cattle-pestivirus challenge.This represents an inter-species transfer of virus and sheep may haveless effective methods of protection from a virus that has crossed thespecies barrier.

TABLE 13 Amount of virus (50% Tissue-Culture Infectious Dose -TCID₅₀) infoetal tissues collected from subunit-vaccinated and control(unprotected) ewes Log₁₀ Pestivirus TCID₅₀ per gram in Foetal TissuesControl (Unprotected) Foetus No. Vaccinated ewes Ewes Number positive7/16 = 44% 8/8 = 100% 1 <2.3 6.7 2 <2.3 5.9 3 4.9 7.0 4 3.0 6.3 5 3.07.4 6 5.4 6.8 7 2.5 3.4 8 Remaining 9 foetuses 6.7 (56%) = No virus Mean(Log₁₀) ± SD 3.34 ± 1.2** (7) 6.2 ± 1.2 (8) **Statistically significantdecrease in viral amount in tissues (P < 0.01)

Notes:

Almost 1000 times less virus in the 7 foetuses from vaccinated ewescompared to the average level in 8 foetuses from unprotected ewes.

14 of the 16 foetuses in subunit-vaccinated ewes either fully protected(9) or with <10³ virus particles per gram of tissue (5 foetuses).Therefore, complete or partial protection from foetal transfer of virusin vaccinated ewes=87%.

A second trial conducted in 24 sheep investigated the protective effectof just one immunogenic protein from cattle pestiviruses. A total of 16sheep, in 2 groups of 8, were vaccinated with 2 different preparationsof the pestivirus envelope glycoprotein, E2, either coupled toheat-shock proteins (hsps) or with no hsps present. A further 8 sheepwere injected with a control preparation that contained neitherpestivirus protein, nor hsps. The trial format was the same as in theprevious trial and the foetal assay results showed that a singlepestivirus subunit glycoprotein incorporated in a vaccine gave only weakprotection against the transfer of the same live BVD virus (Glen Innes)into the foetus. Overall, there was only 29% protection in thevaccinated ewes with the other 71% of foetuses infected. However, it isnoteworthy that the vaccine where the E2 glycoprotein was associatedwith hsps gave almost twice the protective effect as the vaccine wherethere were no hsps. While the result was not significant due to the lownumbers of protected foetuses involved, the trend shows that hsps dohave an effect in enhancing vaccine efficacy. There was, again, a 100%infection rate ( 7/7) in the control ewes. This trial clearlydemonstrated that more than one protein from pestiviruses is required togive high levels of protection. It is clear from both the cattle andsheep trials that a critical component of the subunit vaccine is thenon-structural protein NS3/NS4A coupled to heat-shock proteins. The roleof the smaller envelope glycoprotein E0 is less clear but has a probablerole in protection as well.

EXAMPLE 5 Multiple-Expression Systems for Pestivirus Proteins

To improve the commercial viability of subunit pestivirus vaccines it isnecessary to express more than one protein in a recombinant baculovirus.This cuts down the time-consuming and expensive culture systems inherentin single-protein expression. In order to achieve the goal of reducing 6different expression systems involved in the original subunitpreparation to a maximum of 2 cultures we investigated the possibilityof genetically-engineering baculovirus multiple expression-vectors.

The commercial Multiple Transfer Plasmid, pBAC4x-1, was purchased fromNovagen (Catalogue no. 70045-3) and 4 different genomic regions of twoBVD viruses were successfully inserted into this plasmid. The E1/E2regions from the Australian viruses Trangie and Bega, together with thetruncated NS3 region from the Trangie virus and a capsid/E0 region fromthe same virus, were inserted in the 4 multiple-restriction sites of theplasmid in the following order such that TC/E0 and BE1/E2 were under thecontrol of the baculovirus p10 promoter and TE1/E2 and TNS3/NS4A wereunder the control of the baculovirus polyhedrin promoter. All 4 genomicregions were subsequently shown to be in the correct orientation, givinga transfer plasmid that contained the correct genetic information for 4different BVDV proteins.

Recombinant baculoviruses were then constructed by transfection usingthe pBAC4x-1 transfer plasmid before being cloned in a series of plaqueassays. The recombinant baculoviruses generated were tested at eachstage of the cloning process to see if they would express all 4 proteinsat high levels in Sf9 insect-cell cultures. One recombinant virus wasshown to have stable, high-level expression of all 4 proteins after 3rounds of plaque purification followed by 3 passes of the cloned virusin Sf9 cultures. The levels of expression for each of the 4 proteinswere assayed by titration using specific monoclonal antibodies. Theresults for protein levels in both supernatant and in cells are shown inTable 14. It can be seen that all 4 proteins were produced in asingle-culture system at very high levels, with the individual titrationend-points in the range of 1 in 64 to 1 in 4096.

TABLE 14 Multiple protein-expressing baculovirus cloned 3 times bylimiting dilution and passed 3 times in Sf9 cells. Detecting AntigenEndpoint Dilution Antibody Trangie and Bega 1 in 4096 E2 mAb mix E2(R1465) (Supernatant) Trangie E0 1 in 256 E0 mAb 15c5 (Supernatant)(R495) Trangie NS3 1 in 64 NS3 mAb mix (Supernatant) (R1526) Trangie andBega 1 in 1024 E2 mAb mix E2 (Cells) (R1465) Trangie E0 1 in 4096 E0 mAb15c5 (Cells) (R495) Trangie NS3 1 in 256 NS3 mAb mix (Cells) (R1526)Recombinant baculovirus stable and able to express all proteins to ahigh level.

To further investigate whether the multiple-expression system would besuitable for vaccine production, the Sf9 cells infected with therecombinant 4×-protein was also subjected to heat shock (43° C. for 10min). Protein production in the culture was followed and the finalyields of protein suitable for incorporation in a vaccine ascertained at4 days. A comparison was made with the levels produced in cultures notsubjected to heat shock. The following Table (15) presents the resultsof assaying protein levels in a harvest of both supernatant and cellsfrom each of the cultures:

TABLE 15 Titration endpoint levels for E2, E0 and NS3 proteins harvestedfrom both supernatant and cells in the multiple-expression system.Expressed Titration endpoint Titration endpoint Pestivirus Protein Nohsps With hsps coupled E1/E2 (Trangie + Bega) 1 in 1024 1 in 2048Capsid/E0 (Trangie) 1 in 1024 1 in 2048 Truncated NS3 (Trangie) 1 in1024 1 in 1024 Cultures were grown at 27.5° C. for 4 days and oneculture subjected to heat-shock to couple the pestivirus proteins tohsps. Comparative levels with/without hsps are shown.

It is noteworthy that protein production in the heat-shocked culturecontaining the multiple-protein expressing recombinant pestivirus wasnot affected by heat-shock treatment. Therefore, the baculovirus doesnot decrease protein production and high levels of proteins coupled tohsps are possible with this system. It is concluded that this system iseminently suitable for vaccine production purposes.

By way of a further example, a second Multiple Transfer Plasmid isconstructed containing cDNA encoding further pestivirus proteins, namelyTrangie E1/E2, Clover Lane E1/E2 and Clover Lane NS3/NS4A and optionallyClover Lane E2/Bega E0. It is considered that these additional proteinswill extend the protective effect of subunit vaccines to cover a widerrange of the antigenic diversity shown by BVDV isolates in the field. Inparticular, the inclusion of the 2 border disease virus (BDV) proteinsfrom the ‘Clover Lane’ isolate will extend the protective effect tomatch that of the original subunit vaccine containing 6 proteins. Inthis way, just 2 cultures subjected to a short period of heat treatmentwill produce 6 different pestivirus proteins coupled to hsps. These canbe tested in cattle for efficacy in protecting against the transfer oflive pestivirus into the developing foetus, in the same way as describedin Example 3.

1-20. (canceled)
 21. A composition comprising an immunogenic complexcomprising a non-mammalian heat shock protein (hsp) coupled to aheterologous antigenic polypeptide produced by the process of: (a)expressing the antigenic polypeptide in a non-mammalian cell which cellhas been subjected to a stimulus which causes the induction of a heatshock response in said cell; and (b) recovering the antigenicpolypeptide coupled to one or more hsps from said cell or the culturemedium; and (c) introducing an acceptable carrier or diluent.
 22. Thecomposition produced by the process of claim 21 wherein the cell is anon-mammalian eukaryotic cell and the hsp is a non-mammalian eukaryotichsp.
 23. The composition produced by the process of claim 22 wherein thecell is an insect cell and the hsp is an insect hsp.
 24. The compositionproduced by the process of claim 23 wherein the antigenic polypeptide isan antigen of a pathogenic organism, or a fragment or derivativethereof.
 25. The composition produced by the process of claim 24 whereinthe pathogenic organism is a virus or a bacterium.
 26. The compositionproduced by the process of claim 25 wherein the virus is a pestivirus.27. The composition produced by the process of claim 26 wherein thevirus is bovine viral diarrhoea virus (BVDV).
 28. The compositionproduced by the process of claim 21 wherein the antigenic polypeptide isexpressed in the cell by the introduction into the cell of apolynucleotide encoding the antigenic polypeptide operably linked to aregulatory control sequence capable of directing expression of thepolypeptide in the cell.
 29. The composition produced by the process ofclaim 28 wherein the polynucleotide is part of a virus or viral vector.30. The composition produced by the process of claim 29 wherein the cellis an insect cell and the virus or viral vector is a baculovirus orbaculovirus vector.
 31. A composition comprising a heat shock protein(hsp) derived from a non-mammalian eukaryote coupled to a heterologousantigenic polypeptide and an acceptable diluent or carrier, wherein thecomposition is capable of inducing an immune response to said antigenicpolypeptide in an animal or human.
 32. A composition according to claim31 wherein the hsp is an insect hsp.
 33. A composition according toclaim 32 wherein the antigenic polypeptide is an antigen of a pathogenicorganism, or a fragment or derivative thereof.
 34. A compositionaccording to claim 33 wherein the pathogenic organism is a virus or abacterium.
 35. A composition according to claim 34 wherein the virus isa pestivirus.
 36. A composition according to claim 35 wherein the virusis bovine viral diarrhoea virus (BVDV).
 37. A composition comprising apestivirus antigen coupled to a heat shock protein.
 38. A method ofinducing immunocompetence in an animal against a pathogen, said methodcomprising the steps of administering to an animal a therapeuticallyeffective amount of a composition according to claim 31.